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ISOLATION AND CHARACTERIZATION OF 22°C CHELATOR-SOLUBLE
PECTIC POLYSACCHARIDES OF Lemna minor

By

Liang Cheng

A DISSERTATION
Submitted to
Michigan State University

in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Biochemistry

1996

ABSTRACT
ISOLATION AND CHARARCTERIZATION OF 22°C CHELATOR-SOLUBLE
PECTIC POLYSACCHARIDES OF Lemna minor
By

Liang Cheng

The methods used to isolate, purify and characterize pectic polysaccharides have
significant limitations. Procedures were developed for the isolation of cell walls and for the
extraction, purification and characterization of pectic polysaccharides. The chelator-soluble
pectic polysaccharides were isolated from cell walls of Lemna minor and characterized.

Procedure were developed for preparing purified cell walls. Chelator-soluble pectic
polysaccharides were solubilized under non-degradative conditions and accounted for 31.7%
(by weight) of the total pectic polysaccharides of the cell walls.

The recovery of pectic polysaccharides from diethylaminoethyl (DEAE) anion-exchange
columns was found to be greatly influenced by cations. Six pectic polysaccharides from
citrus, apple, duckweed, and celery were quantitatively (or nearly so) eluted from DEAE-
Trisacryl columns by 0.5 M NH4C1 in ammonium oxalate buffer. Li+ and Cs+ are as effective
as NH; but the presence of Na“ or K+ resulted low sample recovery. A procedure for
purifying pectic polysaccharides from cell walls was developed.

Homogeneity of polysaccharides with respect to sugar composition, molecular size and
methylester content was established by analyzing the polysaccharide material in individual

DEAE column fractions with micro-scale methods. Laser light scattering was used with high

performance anion exchange chromatography to determine true MW. Two chemically
homogeneous pectic polysaccharides, PS-IIb and PS-IVb, were isolated from L. minor.

PS-IIb is an apiogalacturonan with a peak MW range of 28,100 - 99,700. It consists of
a backbone of cc-l,4-linked D-galacturonic acid units with l,3'-linked apiobiose as the
predominant side chains attached to the 0-2 positions of approximately every other
galacturonosyl residues. Terminal-linked rhamnose (2-3%), xylose (2.5%), arabinose (1.4%)
and trace amounts (<O.5%) of fucose, mannose, and glucose were detected in PS-IIb.

PS-IVb is as an a-l,4-linked homogalacturonan with a peak l\7lw range of 53,300 to
2163,000. PS-IVb consists of 96.3% non-esterified galacturonic acid units, apiose (2.1%),
and xylose (1.6%). Trace amounts of rhamnose, fucose, arabinose, mannose, and glucose
were detected in PS-IVb. PS-IVb was virtually completely degraded by poly-[1,4-a-D-
galacturonide] glycanohydrolase (EC 3.2.1 .15).

A micro-scale method for depolymerizing pectic polysaccharides was developed.

To my parents, my wife and my son

iv

ACKNOWLEDGMENTS

I am truly grateful to my graduate advisor and mentor, Dr. Paul K. Kindel, for
offering me an opportunity to work with him, for his guidance, support, and patience, and
for being always willing to help me whenever I needed. I was benefited tremendously from
his advice, both in science and in general, over the years.

I thank the members of my committee, Dr. Rawle I. Hollingsworth, Dr. Estelle
McGroarty, Dr. Jack Preiss and Dr. Robert Bandurski, for their guidance and valuable
suggestions.

A special thanks goes to Dr. Hollingsworth for allowing us to have weekly joint-
group meetings in the past three years and for his generous advice on carbohydrate chemistry.
Thanks also go to all members in Dr. Hollingsworth’s group for their friendship and helpful
discussions.

I thank Dr. Douglas Gage and Ms. Bev Chamberlin of Mass Spectrometry Facility
for their assistance. I am grateful for Dr. Gage’s participation in my final examination in
defense of the dissertation. I also owe thanks to Dr. Long D. Le of NMR Facility for his
technical assistance.

Thanks to all my Chinese friends in MSU, especially in Biochemistry department,
who shared wonderful times with me when cooking and enjoying Chinese food, playing with
computer and chatting together. You made the years in Lansing full of fun.

Finally, I am most grateful to my parents for their unconditional love, consistent
support and faith in me, and to my wife and son, whose love and full cooperation are the

endless source of my energy.

TABLE OF CONTENTS

Page
LIST OF TABLES ...................................................... xi
LIST OF FIGURES .................................................... xii
LIST OF ABBREVIATIONS ............................................ xiv
CHAPTER 1: LITERATURE REVIEW .................................. 1
INTRODUCTION .............................. ' ..................... 2
LITERATURE REVIEW ............................................. 4
Primary cell wall of plants ........................................ 4
Types of pectic polysaccharides .................................... 9
Homogalacturonan ......................................... 10
Rhamnogalacturonan-I ...................................... 1 l
Rhamnogalacturonan-II ..................................... 14
ApiogaIacturonan .......................................... 14
Properties of pectic polysaccharides ................................ l6
Interaction of pectic polysaccharides — formation
of junction zones, egg box structures and gels ................ 16
Stability of pectic polysaccharides ............................. 17
Fractionation and purification of plant cell wall pectic
polysaccharides and determination of homogeneity ............... 18
OBJECTIVE ...................................................... 21
REFERENCES .................................................... 23
CHAPTER II: ISOLATION OF CELL WALLS AND SOLUBILIZATION
OF PECTIC POLYSACCHARIDES FROM Lemna minor ..... 30
ABSTRACT ...................................................... 31

vi

INTRODUCTION .................................................. 32

MATERIALS AND METHODS ...................................... 34

Materials and general methods .................................... 34

Preparation of cell walls ................................ 34

Solubilization of pectic polysaccharides from cell walls ............ 35

Fractionation of P-SDC and P-NaCl on DEAE-Sephadex ........... 36

Size exclusion column chromatography ............................. 37

RESULTS ........................................................ 37
Comparison of cell walls disrupted with

the Waring blender and French press ........................... 37

Media used for cell wall homogenization ............................ 37

Sequential extraction of pectic polysaccharides from cell walls ........... 40

DISCUSSION ..................................................... 45

REFERENCES .................................................... 47

CHAPTER III: EFFECT OF CATIONS ON THE ELUTION
OF PECTIC POLYSACCHARIDES FROM

ANION EXCHANGE RESINS ........................... 49
ABSTRACT ...................................................... 50
INTRODUCTION .................................................. 51
MATERIALS AND METHODS ...................................... 52

Materials and general methods .................................... 52
Analytical anion exchange column
chromatography of pectic polysaccharides ....................... 53
Preparative anion exchange column chromatography
of a pectic polysaccharide fraction from L. minor ................. 55
Effect of salts on the solubility of apple pectic acid .................... 55
RESULTS ........................................................ 56
DISCUSSION ..................................................... 65
REFERENCES .................................................... 69

vii

CHAPTER IV: A MICRO-SCALE METHOD FOR DEPOLYMERIZATION

OF PECTIC POLYSACCHARIDES ...................... 71

ABSTRACT ...................................................... 72
INTRODUCTION .................................................. 73
MATERIALS AND METHODS ...................................... 74
Materials ..................................................... 74
Depolymerization of pectic polysaccharides .................... 75
Establishing conditions for Method 111 ....................... 76
HPAEC-PAD ....................................... 76
Standard sugar mixture treated by Method 111 .................. 77
RESULTS ........................................................ 77
Comparison of Methods 1, II and III ........................ 77
Optimization of conditions for Method 111 ..................... 78
Standard sugars mixture treated with Method HI ................. 80
DISCUSSION ..................................................... 86
REFERENCES .................................................... 89

CHAPTER V: HOMOGENEITY AND STRUCTURE OF THE

22°C AMMONIUM' OXALATE-SOLUBLE

PECTIC POLYSACCHARIDES OF Lemna minor ............ 91
ABSTRACT ...................................................... 92
INTRODUCTION .................................................. 93
MATERIALS AND METHODS ...................................... 94

General method ................................................ 94
Isolation of pectic polysaccharides ................................. 95
Rechromatography of pectic polysaccharides

with DEAE-Trisacryl Plus M ................................. 95
Refractive index increment (dn/dc) of polysaccharide samples ........... 96
Determination of MW and HPSEC .................................. 99
Depolymerization of pectic polysaccharides

and preparation of alditol acetates ............................ 104

viii

Gas chromatography and mass spectrometry ........................ 104

Determination of degree of methyl esterification ..................... 105
IH NMR spectroscopy .......................................... 105
RESULTS ....................................................... 106
Isolation of pectic polysaccharides ................................ 106
MW, Mn and Polydispersity ...................................... 106
HPSEC ..................................................... 113
Sugar composition of polysaccharides ............................. 115
Degree of methyl esterification ................................... 118
1H NMR spectroscopy .......................................... 118
Rechromatography of pectic polysaccharides ........................ 121
DISCUSSION .................................................... 121
REFERENCES ................................................... 128
CHAPTER VI: STRUCTURAL ANALYSIS OF PECTIC
POLYSACCHARIDES PS-IIb AND PS-IVb ............... 130
ABSTRACT ..................................................... 131
INTRODUCTION ................................................. 132
MATERIALS AND METHODS ..................................... 134
General method ............................................... 134
Materials .................................................... 134
Carboxyl reduction ............................................ 134
Methylation .................................................. 134
Hydrolysis, reduction and acetylation .............................. 135
GC-MS determination of the partial methylated alditol acetates ......... 136
Pectinase degradation of polysaccharides PS-IIb and PS-IVb ........... 136
High performance anion exchange chromatography
with pulse amperometric detection ..................... 137
RESULTS ....................................................... 138
Methylation analysis .................................. 138
Enzyme degradation of polysaccharides PS-IIb and PS-IVb ............ 146
DISCUSSION .................................................... 152

ix

REFERENCES ................................................... 155

CHAPTER VII: CONCLUSIONS AND FUTURE PERSPECTIVE ......... 158
CONCLUSIONS .................................................. 1 59
FUTURE PERSPECTIVE ........................................... 162
REFERENCES ................................................... 165

APPENDIX A: SOLUBILIZATION OF PECTIC POLYSACCHARIDES
FROM THE CELL WALLS OF Lemna minor AND
Apium Graveolens ..................................... 166

APPENDIX B: REEXAMINATION OF THE ACETYLATION OF
APIITOL IN THE DETERMINATION OF APIOSE ........ 172

APPENDIX C: DERIVATION OF EQUATIONS FOR CALCULATION
OF THE AMOUNT OF NEUTRAL SUGAR AND
URONIC ACID IN SAMPLES ........................... 177

APPENDIX D: A COMPUTER PROGRAM FOR CALCULATION
OF THE AMOUNT OF NEUTRAL SUGAR AND
URONIC ACID IN SAMPLES ........................... 183

APPENDIX E: DERIVATION OF THE EQUATION USED TO
CALCULATE THE GALACTITOL ACETATE-D2
IN GALACTITOL ACETATE ........................... 190

Table 2.1.

Table 2.2.

Table 3.1.

Table 3.2.

Table 3.3.

Table 4.1.

Table 4.2.

Table 5.1.

Table 5.2.

Table 6.1.

LIST OF TABLES

Pectic substances solubilized, total cell wall protein and relative
molecular weight of the pectic polysaccharides from cell walls

prepared in different homogenizing media ...................... 41

Solubilization of pectic polysaccharide

from purified cell walls of L. minor ............................ 44

Effect of Cations on the Recovery of Apple Pectic Acid

from DEAE-Trisacryl Plus M and DEAE-Sephadex A-25 .......... 57
Effect of Cations on the Recovery of Different

Pectic Polysaccharides from Analytical Columns

of DEAE-Trisacryl Plus M .................................. 59
Effect of salt and resin types on the elution of the

22°C ammonium oxalate-soluble pectic polysaccharide

fraction of L. minor from ion-exchange columns ................. 61
Products detected after depolymerization of

three pectic polysaccharides by Methods I - III .................. 79
Optimization of conditions for depolymerization by Method III . . . 81
Refractive index increment of pectic polysaccharides ............. 100
The MW, MW and polydispersity (MW/Mn) of selected

pectic polysaccharide samples from the DEAE column

chromatogram of Figure 5.3 ................................. 109

Glycosyl-linkage composition (mol%) of

PS-IIb and PS-IVb from methylation analysis ................... 147

xi

Figure 1.1.

Figure 1.2.
Figure 1.3.
Figure 2.1.

Figure 2.2.

Figure 3.1.

Figure 4.1.

Figure 4.2.

Figure 5.1.

Figure 5.2.

Figure 5.3.

Figure 5.4.

Figure 5.5.

Figure 5.6.

LIST OF FIGURES

Proposed primary cell wall of most

flowering plants, designated type I .............................. 5
The proposed type 11 cell wall .................................. 7
Diverse structures of the pectic polysaccharides .................. 12
Light microscopic view of cell walls of L. minor .................. 38

Column chromatograms of L. minor cell wall pectic
polysaccharide fractions P-SDC (a) and P-NaCl (b) ............... 42

Precipitation of apple pectic acid with
NH4C1, NaCl, KC], CsCl and LiCI ............................ 63

Galacturonic acid remaining after depolymerization of apple
pectic acid by Method 111 used with varying methanolysis times ..... 82

GC response factors of eight sugar alditol
peracetates and myo—inositol peracetate
(as internal standard) based on area/mg of sample ................ 85

Relationship between RRI reading or calculated refractive
index at 950 nm (n—no)950 um and concentration of N aCl ............ 97

Relationship between Km, and Log M“, for those pectic
polysaccharide fractions analyzed by HPSEC and MALLS ......... 102

Column chromatogram of the 22°C ammonium
oxalate-soluble fraction from purified cell walls of L. minor ........ 107

HPSEC column chromatograms of L. minor
pectic polysaccharides F217 (a) and F270 (b) ................... 111

Sugar composition of the pectic polysaccharide(s)
in individual column fractions from the DEAE-

Trisacryl Plus M column chromatography (Figure 5 .3) ............ 116

The low—field region of the 500 MHz 1H NMR spectra of L. minor

xii

Figure 5.7.

Figure 6.1.

Figure 6.2.

Figure 6.3.

Figure 6.4.

Figure 6.5.

pectic polysaccharide F289 (a) and polygalacturonic acid (b) ....... 119
Column chromatograms of PS-IIb and PS-IVb .................. 122

Gas-liquid chromatograms of partial methylated
alditol acetates derived from pectic polysaccharides
PS-IIb (a) and PS-IVb (b) ................................... 139

E.i. mass spectrum and proposed fragmentations
of 1 ,4-di-0-acetyl-( 1 -deuterio-)-2,3,3'-tri-0-methyl
apiitol (t-Api, peak 2 of Figure 6.1a) .......................... 142

E.i. mass spectrum and proposed fragmentations
of 1 ,3',4-tri-O-acetyl-(1-deuterio-)-2,3-di-0-methyl
apiitol (3'-Api, peak 6 of Figure 6.1a ) ......................... 144

Column chromatograms from HPAEC-PAD for
polysaccharide PS-IIb after various treatments ................... 148

Column chromatograms from HPAEC-PAD for
polysaccharide PS—IVb after various treatments ................. 150

xiii

Api
Ara
AUA
BCA
BSA
CDTA
DE
DEAE
DM
DMSO

EDAC

Fuc

Gal
GalA
GC
GC-MS

Glc

HPAEC-PAD High performance anion exchange chromatography - pulse amperometric

LIST OF ABBREVIATIONS

Apiose

Arabinose

Anhydrogalacturonic acid
Bicinchoninic acid

Bovine serum albumin
1,2-Cyclohexanediaminetetraacetic acid
Degree of esterification
Diethylaminoethyl

Degree of methylesterification
Dimethyl sulfoxide
1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide
Flame ionization detector

Furanose

Fucose

Galactose

Galacturonic acid

Gas chromatography

Gas chromatography-mass spectrometry

Glucose

xiv

HPLC
HPSEC
MALLS
Man
MS
NMR
PC
PGUA

PMAA

Rha
RG-I

RG-II

SDC

SDS

TFA

Xyl

detection

High performance liquid chromatography
High pressure size exclusion chromatography
Multi-angle laser light scattering
Mannose

Mass spectrometry

Nuclear magnetic resonance

Personal computer

Polygalacturonic acid

Partially methylated alditol acetate
Pyranose

Rhamnose

Rhamnogalacturonan-I
Rhamnogalacturonan-II

Relative refractive index

Sodium deoxycholate

Sodium dodecyl sulfate

Terminal-

Trifluoroacetic acid

Xylose

XV

CHAPTER I

LITERATURE REVIEW

2

INTRODUCTION

Cell walls of growing plants provide mechanical support and a protective barrier for
cells. They are also dynamic in their shape, composition and properties in response to
growth, stage of differentiation, environment, pathogen invasion, and their activities
including signal conducting and regulating enzyme activity. If we can alter the composition
and the structure of plant cell walls, it may be possible to make them more resistant to
pathogenic and environmental stress. It also may be possible to improve them as a clean,
economical, and renewable source of biomass for fuel and other chemicals. But before we
can alter the plant cell walls through molecular biological techniques, we have to know how
the cell wall polysaccharides are synthesized and this in turn requires that their structure be
known. Our knowledge about plant cell walls and their constituent polysaccharides lags
behind that of proteins and nucleic acids due to: (i) technical difficulties in isolating “intact”
polysaccharides from cell walls, (ii) the heterogeneous nature (both in size and composition)
of the polysaccharides, and (iii) the fact that the sugar sequence of cell wall polysaccharide
is not directly specified by a template. Much of the current research on plant cell walls is
still based on identifying and elucidating the covalent structures of the macromolecular
components of the walls.

Pectic polysaccharides are present in all higher plants. They are found in the primary
cell wall, usually as the most abundant component. Pectic polysaccharides are extracted
from the middle lamella and primary cell wall with hot water, chelating agents, dilute alkali,

. weak acids and enzymes such as endo-l ,4—a-polygalacturonase (EC 3.2.1.15) (1-7). Three

3

types of pectic polysaccharides or pectic polysaccharide units, homogalacturonan, RG-I and
RG-II, have been isolated from a number of different plants and characterized (8,9). Pectic
polysaccharides of the cell wall of Lemna minor have a somewhat different sugar
composition than the pectic polysaccharides from other plants in that apiose is the main
neutral sugar (IO-13). Isolation and characterization of pectic polysaccharides from other
plants with apiose as a major component has not been reported, although apiose is widely
distributed in higher plants (14) and results suggest it is present in polysaccharides (15,16).

In this review, recent models of primary cell walls of plants are discussed. The major
types of known pectic polysaccharides are reviewed including the partially characterized,
apiose—containing pectic polysaccharides of L. minor. Physical and chemical properties of
pectic polysaccharides related to their solubilization, purification and function in cell walls
are examined. Current problems in fractionation, purification and homogeneity of pectic
polysaccharides are discussed. Literature reviewed in this chapter will provide us with a
general understanding of cell wall pectic polysaccharides. Issues related to each specific

chapter are reviewed in the Introduction of that chapter.

4

LITERATURE REVIEW

Primary cell wall of plants

Primary cell walls of plants are highly hydrated. On a dry weight basis, they consist of
~90% polysaccharides and ~ 10% proteins (9,17). Recently, models for the primary cell
walls of flowering plants (type I cell wall) and the Poaceae (type 11 cell wall) were proposed
by Carpita and Gibeaut (18) based on the structures of known polysaccharides and the
physical properties of cell walls. In their model, the type I cell wall has a skeleton of
cellulose microfibrils (Figure 1.1a) which is interlaced and interlocked by xyloglucan
polymers (Figure 1.1b) through hydrogen bounding and the cellulose microfibrils-xyloglucan
framework (about 50% of the wall mass) is embedded in a matrix of pectic polysaccharides
(about 30% of the wall mass). The type H cell wall is also composed of a cellulose
microfibrils network (Figure 1.2a) but it is interlocked by glucuronoarabinoxylans (Figure
1.2b) instead of xyloglucans. A smaller amount of pectic polysaccharide is found in the type
H primary cell wall (19) and large portions of the non-cellulosic polymers are “wired on”
(18) the microfibrils by phenolic cross-links (Figure 1.2a). In both type I and type 11 cell
walls, the major pectic polysaccharides proposed are polygalacturonic acid and
rhamnogalacturonan (RG); the latter are substituted with polysaccharide side chains of
arabinan, galactan and arabinogalactan. Adjacent pectic polysaccharide chains can associate
to form junction zones through their free carboxyl groups and Ca2+ bridges (20-22). Pores
are formed when the junction zones are separated and spaced by the degree of esterification

. and the side chain-bearing regions of the pectic polysaccharides. It has been suggested that

Figure 1.1. Proposed primary cell wall of most flowering plants, designated type 1.
Taken from reference (18). (a) Representation of a single stratum of the cell wall just after
formation in dividing cells of a meristem. Several strata such as this coalesce to form a wall
(see text for a further description of the wall). (b) A unit structure of xyloglucan in type I cell
walls. The basic heptasaccharide repeating unit structure may bear additional substitutions

as indicated.

 

 

 

 

 

 

PGA Junction
zone

4
4?}
3 RG lwnh
- garabinogalactan
X . fidechams
5‘. A __\,'

 

 

Figure 1.2. The proposed type II cell wall. Taken from reference (18). (a) Representation
of a single stratum of microfibrils just after cell division. The microfibrils are interlocked
by glucuronoarabinoxylans (GAX). A substantial portion of the non-cellulosic polymers are
‘wired on’ the microfibrils by alkali-resistent phenolic linkages. (b) A unit structure of the

highly substituted glucuronoarabinoxylan.

 

 

 

 

 

 

W 74

RG I with arabino-
galactan side-chains

 

Xonglucan

 

 

 

 

 

 

 

 

Phenolic cross-links

 

PGA junction zone

 

9

the expansion of the cell wall during growth is modulated by pectic polysaccharides through
pore size (barrier or opening to enzymes) (23), which, in turn, is affected by degree of
methylesterification (18), availability of Ca2+ ions (24), and local pH (in vicinity of
xyloglucan) (18). One mechanism proposed for controlling growth was that the pectin
methylesterase located in the cell wall controlled the pH by cleaving the ester linkage of the
uronate esters and converting them to free acids (25,26). The esterification of galacturonosyl
residues in pectic polysaccharides of maize embryonal coleOptiles was found to correlate
with the elongation of the cell wall (27). Extensin, the hydroxyproline-rich glycoprotein
(28), also may play an important role in the mechanism of locking the cell wall into a
particular shape after elongation; the microfibrils arranged in the transverse axis are cross-
linked by xyloglucans in the longitudinal axis and by extensins oriented radially, “like pins
stuck into thick cloth” (28). The details of the structure of primary cell walls and our
knowledge about its metabolism are still far from complete. Since the cell wall models are
based heavily on the structure of known polysaccharides, the isolation and characterization

of additional polysaccharides will greatly improve our knowledge about plant cell walls.

Types of pectic polysaccharides

Pectic polysaccharides are a family of acidic polysaccharides with a 1,4-linked a—D-
galactosyluronic acid-rich backbone which may be interspersed with 1,2-linked a-L-
rhamnosyl residues, and have neutral sugar side chains attached. The carboxyl groups of the
galacturonosyl residues may be free or partially or fully methyl esterified (8). Pectic

_ polysaccharides without or with only a negligible content of methyl ester groups are pectic

10

acids, and those with “various, but greater than negligible, contents of methyl ester groups”
are pectins (29). Plant cell wall pectic polysaccharides have been classified as one of three
types: homogalacturonan, rhaminogalacturonan I and rhaminogalacturonan H.
Homogalacturonan. Homogalacturonans are defined as polymers consisting solely
or “predominantly” (9) (i.e. containing ~90% or higher GalA) of a-1,4-linked-D-
galacturonosyl residues. A homogalacturonan with 3% galactose was isolated from
suspension-cultured Rosa cell walls by sequential treatment with 2.5 M NaOH, 1.0 M HZSO4
at 120°C, and 0.15 M NaOH (30). This polymer was not purified by column
chromatography (homogeneity was not determined) and the molecular size and degree of
esterification are unknown. Two homogalacturonans containing 93.9% and 92.7%
galacturonic acid were solubilized by hot water from Zea shoots (31) but their degree of
esterification was not determined. A pectic polysaccharide containing 91% galacturonosyl
residues and with a relative Mw<10,000 (relative to dextran) was purified from rice
endosperm cell walls that were treated with glucoamylase and protease and extracted with
0.25% ammonium oxalate at 90°C for 24 h (32); the degree of esterification was not
determined. This suggests the possible presence of homogalacturonans in monocots.
Homogalacturonans were also found in the media of tobacco (87% galacturonic acid) (33)
and sycamore (95% galacturonic acid) cell suspension cultures (9). Recently,
homogalacturonan fractions with > 98% galacturonic acid residues and degrees of
polymerization of 72- 100 were isolated from de-esterified pectic substances of citrus (DM
= 72), apple (DM = 75) and sugar beet (DM = 54) with 0.1 M HCl at 80°C for 72 h (34).

, However, no pure homogalacturonan has been isolated from primary cell walls without using

11

treatments that are likely to cleave covalent bonds. In addition, the size homogeneity of
isolated homogalacturonans has not been established.

Rhamnogalacturonan-I. RG-I, the pectic polysaccharide first isolated from the cell
walls of suspension-cultured sycamore cells after the treatment of cells with endo-a-1,4-
polygalacturonase, has been studied in some detail (35-39). With a molecular weight of
about 200,000 (estimated by comparing the mobility of RG-I with that of globular proteins
and dextrans on an agarose A-Sm column), the backbone of RG—I consists of chains of a— 1 ,4
linked D-galacturonosyl residues interspersed with a—1,2-linked L-rhamnosyl residues (Figure
1.3b). Approximately half of the 1,2-linked rhamnosyl residues are branched and at least 30
different side chains have been found attached to O-4 of the rhamnosyl residues (9). The
side chains are rich in L-arabinosyl and D-galactosyl residues, although small amounts of
other sugars, such as D-xylose and L-fucose, were also found (35,40). Three major types of
side chains are arabinans, type I arabinogalactans and type II arabinogalactans (Figure 1.3c)
( 18). Glucuronic acid and 4-0—methyl-glucuronic acid residues were found to be
components of the galactosyl-containing side chains of RG-I (39). The distribution of the
rhamnosyl residues (with attached side chains) in the backbone is not even. Both regions
free of rhamnosyl residues and side chains, “smooth regions”, and the regions with a high
frequency of side chain-attached rhamnosyl residues, “hairy regions”, were found. The
backbone of “smooth regions”, corresponding to 25 and >72-100 galacturonosyl residues,
were isolated from cell walls of citrus, apple, sunflower (41) and sugar-beet (34) with mild
acid hydrolysis. Fragments of “hairy regions” of the RG-I backbone, with molar ratios of

_ rhamnosyl to galacturonosyl residues of 1-2.3:6.7 (total neutral sugar composition is 79-

12

Figure 1.3. Diverse structures of the pectic polysaccharides. From reference (18). (a)
An example of ‘eggbox’ junction zone. Anti-parallel chains of polygalacturonic acid cross-
linked at unestrified regions by Ca2+ bridges. (b) A section of RG-I backbone. Galacturonic
acid residues (grey shadded) are interspersed with 1,2-1inked rhamnosyl residues. Arabinan
and arabinogalactan side chains are attached to the 4-0 position of the rhamnose. (c)Three

types of side chains that attached to about half of the rhamnosyl residues of RG-I.

   

ze- {Hus-140

   

Y
. - -L-an/—(1—+5)a-L-araf-(I —-)5)a-L-araf-(I 490-0311“ 1 45)-

in" :3:

I T
i i

.—_ Arabinan: '3 E
M

I

Y

‘2‘

.‘i

Arablnans 6

---o-gaip-(|axingawi—Mfio-pwl—Mmplp-uanon-morn»;
A A

a. L uni-(143
a-L-araf-(I -+3

.+—.Anbinogalacums Typo I Arablnogalactnns

WAD-91H14315-DM1*3W143W143)5-01flp4 l 43)B-D-:llP-(l-*3)I3-D-919-(l-+3)
A A

46}

a-L-ml—(I—flyé G'L-W-(l-fl}

)Q-o-gup-(i—aé

a—l/Iflf-(l-fl)‘ mural-(HS)-

a-L-IIIH 1 40543-81“! “’6

a—L-Inf-(l 43)-

a-L-mf-(l-térb-D-ulp-(l-fifi-D-

atwers-wn-mnnw—m

Type II Anblnogaloctnno

14

83%)(42), l:l.5—2.9 (total neutral sugar composition is 30-60%) (34) and 1236-48 (total
neutral sugar composition is 72-82%) (43) were isolated from cell walls of sugar-beet, apple,
citrus and lemon peel with endopolygalacturonase (42), HCl hydrolysis (34) and “Rapidase
C600" (Gist Brocades, Deft, The Netherlands) (43,44). The “hairy region” of RG-I was
found to be degraded by rhamnogalacturonase (44,45). Pectic polysaccharides of the RG-I
type have been found widely distributed in plants, both monocots and dicots, such as tobacco
(46), potato (47), onion (48), alfalfa (49), pea (50), maize and rice (51,52).
Rhamnogalacturonan II. RG-II is another pectic polysaccharide originally isolated
from suspension—cultured sycamore cells after the treatment of cells with endo-a-1,4-
polygalacturonase (6). RG-II is much smaller than RG-I (molecular weight ~11,000), and
has a higher proportion of rhamnosyl residues, which are 3-, 3,4-, 2,3,4- and terminally-
linked instead of 2- and 2,4-linked as in RG-I. In addition, RG-II has many unusual glycosyl
residues including 2-0-methylfucosyl, 2-0-methylxylosyl, apiosyl and 3-C—carboxy1-5-
deoxy-L-xylosyl (aceric acid) residues (53,54). Pectic polysaccharides of the RG-II type are
found in the primary cell walls of other plants such as pea, pinto bean, tomato (6), potato
(47), onion (48), and rice (51,55) which suggests that RG-II type pectic polysaccharides are
common in plant cell walls. Since RG-II is solubilized from primary cell walls with either
endo-ct-l ,4-polygalacturonase or strong base and is usually present in small quantities, it may
be covalently linked as a large side chain to a larger cell wall polysaccharide.
ApiogaIacturonan. Pectic polysaccharides isolated from cell walls of L. minor with
0.5% ammonium oxalate were characterized as apiogalacturonan (10—12). Apiose accounted

. for 25.2-27.9% (10) and 79-38. 1% (11) of the apiogalacturonan and was the major neutral

15

sugar component. The results showed that apiogalacuronan are composed of a linear
galacturonan backbone with D-apiose and apiobiose side chains (11,12). Homogeneity with
respect to molecular size and sugar composition was not determined for apiogalacturonans.
Apiose is present in small amounts in RG-II (6,54) and appears to be present in more
substantial amounts in polysaccharides from Zosteraceae (15,16) and Lemna gibba (56).
Evidence has been presented that apiose is also a constituent of polysaccharides in Posidonia
australis and Tilia sp. (57). Duff (14) reported that of 175 plant species examined, 31
showed "traces" amounts, 51 had "moderate" amounts and 17 were "good sources" of apiose.
Although Duff did not distinguish between apiose in glycosides and apiose in
polysaccharides, his findings showed that apiose is relatively widely distributed in the plant
kingdom. Also, the apiose content of samples possibly is being underestimated by
researchers who determine sugar composition by GC-MS analysis of alditol acetates, since
apiitol and xylitol peracetates have almost identical GC retention times and mass spectra
(6,58).

When apiogalacturonans were isolated from L. minor a number years ago (10- 12), paper
chromatography was used to identify apiose. Results from GC-MS reported later showed
small amounts of rhamnose, xylose and several other neutral sugars are present in these
apiogalacturonans (13). In the earlier work, the homogeneity of these polysaccharides was
not determined. Also the molecular size of the polysaccharides and the position of

attachment of the side chains to the backbone were not determined.

16

Properties of pectic polysaccharides

Interaction of pectic polysaccharides — formation of junction zones, egg box structures
and gels. The apparent pKa of the carboxyl groups of polygalacturonic acid is 4.10 (59).
Esterification of carboxyl groups in the polysaccharide lowers the pK,; the apparent pKa of
a pectin with a DE of 65% is 3.55 (59). In living plant cells most of the unesterified
carboxyl groups of the uronic acid residues of pectic polysaccharides are deprotonated and
usually interact with cations such as Ca2+. In aqueous solution, the polygalacturonic acid
portion of the backbone folds into a 2l-helix conformation (2 stands for two galacturonosyl
residues per conformational repeat unit; the subscript 1 stands for one turn of the helix per
repeat unit)(22,60,61). With formation of ionic and hydrogen bonding, the helical chains
of the polygalacturonic acid portion of the backbones can interact with each other
interrnolecularly to form “junction zones”(62-65). A junction zone is the complex formed
when the portions of two or more anti-parallel helical chains are cross-linked by Ca“. Their
structure has been postulated to be like a “egg box” (Figure 1.3 a) (21,41, 61). The number
of contiguous unesterified uronic acid residues needed to form stable junction zones and the
extent to which several chains can stack together remain unknown both in vivo or in vitro.
Results of statistical calculations suggest that at low Ca2+ concentration, a minimum of 14
galacturonic acid units from each chain are needed to from a stable junction zone (21,41).
If sufficient Ca2+ is present, some interrupting methyl esterified galacturonic acid residues
can be tolerated in the stable junction zone. When excess Ca2+ is available, multiple
polygalacturonic acid chains can stack together to form a calcium pectate gel (21,66). The

. 1,2-linked rhamnosyl residues in ROI interrupt the junction zones by forming “kinks” in the

17

polysaccharide backbone (21,41). Side chains attached to the backbone also prevent
formation of the junction zones. It was suggested that pectic polysaccharides in some species
can be cross-linked to each other and to other non-cellulosic polysaccharides by ester
linkages involving dihydroxylcinnamic acid derivative such as diferulic acid (67).

At sufficiently low pH and in the presence of sufficient sugar, pectic polysaccharides can
form another type of gel, the “acid gel”, without participation of Ca2+. The junction zones
in the acid gel form when the number of charges on the polygalacturonic acid backbone are
reduced enough so that sufficient interchain hydrogen bonds are formed (29). Pectins with
higher DE have less charge so they can form a gel at a higher pH. At constant pH, the gel
strength of an acid gel increases with increasing DB of the pectin. Increasing the
concentration of a monovalent cation, such as N a“ and K*, will result in decreasing hydrogen
bonding in junction zones and thereby decreasing gel strength and reducing precipitation, and
as a result a "salting in" occurs, although too high a concentration of monovalent cations may
cause precipitation (68).

Stability of pectic polysaccharides. The glycosidic linkage of unesterified uronic acid
residues in pectic polysaccharides is quite resistant to acid hydrolysis (69). Esterified or
partial esterified polysaccharides are most stable at about pH 4 (29). Lower pH causes
hydrolysis of some acid-labile glycosidic bonds such as as those involving L-
rhamnopyranosyl (29,70), L—arabinofuranosyl (69) and D-apiosyl residues (12) in pectic
polysaccharides. At pH 4.5, apiose and apiobiose side chains can be completely released
from apiogalacturonans in 3 h at 100°C (12). At pH values of 5-6, pectins are stable only

at room temperature. At high pH, B-elimination causes depolymerization of pectin (71,72).

18

Pectin breaks down rapidly at pH 6.8 with elevated temperature (73,74) through [3-
elimination (74). Since the stability or solubility or both of pectic polysaccharides in solution
change with changes in concentration of polysaccharide, pH, and concentration and type of
cation, great care must be taken to avoid degradation, precipitation and gel-formation

throughout the isolation, purification and characterization processes.

Fractionation and purification of plant cell wall pectic polysaccharides and
determination of homogeneity

Generally, a portion of the pectic polysaccharides of plant cell walls are extracted by
hot water, dimethyl sulfoxide (DMSO) (51), enzymes such as endo-or-l,4-polygalacturonase
(37), chelating agents such as ammonium oxalate, EDTA, and CDTA and weak base such
as Na2CO3 (51). Pectic polysaccharide fractions that were solubilized from cell walls by
sequential extraction with different reagents, such as hot water, ammonium oxalate, dilute
acid and alkali solution, in some cases were directly characterized (sugar composition and
glycosidic linkage) without further purification and without establishing homogeneity
(4,42,43,75). In these cases, there is a possibility that different polysaccharides could be
extracted together in a single extraction and then would contaminate each other. Usually
pectic polysaccharides with different sugar compositions and sizes are co-solubilized with
an individual extractant (4,6,27,35,46,49,76-84) and they need to be purified further before
characterization. In addition, other polysaccharides such as arabinans (85), galactans (86)
and arabinogalactans (87) can be co-solubilized with pectic polysaccharides even when mild

. non~degradative conditions (e. g. chelating agents) are used. It is important to establish the

19

homogeneity of the polysaccharides to be characterized in order to obtain accurate structural
information. Commonly, purification of extracted cell wall polysaccharide involves the use
of anion-exchange chromatography or size exclusion chromatography or both. In some
cases, extracted polysaccharides were pre-fractionationed by precipitation with (NH4)2SO4
(31), ethanol or Cetavlon (cetyltrimethylammonium bromide) (82,83) before being applied
to columns. Pectic polysaccharides have been purified with anion exchange material such
as DEAE-cellulose (4,15,32,46,50,88-90), DEAE-Sephacel (76,77), DEAE-Sephadex
(6,11,27,31,35,76,78,82,91,92), DEAE-Sepharose CL—6B (83,84,93), DEAE-Spectra/Gel M
(49), and DEAE-Trisacryl (5,94,95). Selective elution of pectic polysaccharides from the
column is effected by stepwise or linear gradient elution with salt solutions or buffers.
Usually, the eluted fractions of pectic polysaccharides were tested for uronic acid or total
sugar or both, and fractions were pooled according to the uronic acid and total sugar peaks.
The pooled fractions were directly characterized (such as sugar composition analysis)
(4,5,27,43,49,82) or characterized after further purification by size exclusion chromatography
with Agarose A-5 (32,35), Sephadex 4B (32), Bio-Gel A 5 m (46), Sepharose CL-2B (4),
Sepharose CL-4B (46), Sepharose CL—6B (4,31,83), Bio-Gel P-10 (6), and Sephacryl S-500
(84). In some cases rechromatographed by anion exchange column (31,77) was performed.
Pectic polysaccharides extracted from plant cell walls were also directly purified with size
exclusion chromatography (43,50,51,81). However, in all the above, except one report by
Yamada’s group (83), fractions were pooled based on the elution profiles of total sugar or
uronic acid after either ion exchange or size exclusion chromatography. But the

. homogeneity of polysaccharides in the fractions was not established before they were pooled.

20

A single uronic acid or total sugar peak on either ion-exchange or size exclusion
chromatography does not prove the polysaccharide material is chemically or physically
homogeneous. For example, a pectic polysaccharide fraction, GR-ZIIa, solubilized from the
root cell walls of Glycyrrhiza uralensis and purified by DEAE-Sepharose CL—6B showed a
single, nearly symmetrical peak of uronic acid (as well as total sugar) on size exclusion
chromatography (Sepharose CL-6B) (83). But analysis of the individual fraction across the
peak with size exclusion HPLC (Asahi-pak GS-S 10 + Gs 320) resulted in detection of five
polysaccharides whose molecular size varied from 2.0 x 10“ to 1.9 x 105 and galacturonic
acid contents varied from 41.8% to 85.2% (83). In another example, a pectic polysaccharide
fraction extracted from purified sugar beet cell walls showed a single uronic acid and total
sugar peak on ion-exchange chromatography (DEAE-Sepharose CL—6B, linear gradient of
sodium succinate 005-] M, pH 4.8), but three peaks were observed when it was fractionated
by Sephacryl-8500 size exclusion chromatography (84). With the development of an
improved micro-scale method of sugar composition analysis (Chapter IV), the development
of a procedure for determining MW on small quantities of polysaccharide by using HPSEC
with MALLS (Chapter V), and the use of a micro-scale methyl ester method (96), the
homogeneity with respect to sugar composition, degree of methyl esterification and MW can
be established for polysaccharide material present in individual fractions from ion-exchange
or size exclusion chromatography. This can undoubtedly help us to decide how to pool
fractions with same type of polysaccharide and obtain pure samples for characterization.

Since a mixture of polysaccharides is often extracted with a single extractant (e.g. a

_ chelating agent), incomplete recovery of the polysaccharides from the purification procedure

21

can result in biased information about the character of the polysaccharides in the cell wall.
Poor recovery of pectic polysaccharides has been observed from many types of ion-exchange
columns (95). Sample recovery of pectic polysaccharide of 51%, 82 to 85%, 0 to 87%, 32
to 88% and 24 to 51% have been reported with columns of DEAE-Trisacryl (l), DEAE-
Cellulose (88,89), DEAE-Sephadex (31,76,91), DEAE-Sephacel (77,78,97-100), DEAE-
Sepharose CL-6B (4,101), respectively. Some pectic polysaccharides adsorb strongly onto
the cellulose resin and require the use of strong reagents, such as urea or 0.5 M NaOH, to
release them (88). Chromatography procedures with high recovery of pectic polysaccharide

using non-degradative conditions need to be developed.

OBJECTIVE

The overall goal of this work is the isolation and characterization of the 22°C chelator-
soluble pectic polysaccharides of cell walls of L. minor. Chapter 11 describes a simple
method for the isolation of cell walls and an improved method for polysaccharide extraction.
Chapter III discusses the effect of cations on the elution of pectic polysaccharides from anion
exchange resins. A quantitative method has been developed for fractionation and elution of
pectic polysaccharides from anion exchange columns. In Chapter IV, an improved micro—
scale method of depolymerization of pectic polysaccharide samples for GC-MS and HPLC
analysis is described. The homogeneity of the purified pectic polysaccharides, with respect
to molecular size, sugar composition and degree of methyl esterification, are examined in

, Chapter V. Chapter VI provides information on the questions: How are the sugar residues

22

in two purified pectic polysaccharides, PS-IIb and PS-IVb, linked? What is the primary
structure of these two polysaccharides? Results of methylation analysis, partial hydrolysis
and enzyme degradation are discussed. In the last chapter, Chapter VII, general conclusions
are made for knowledge gained in the previous chapters and directions for future research

are suggested.

10.

11.

12.

13.

, 14.

23

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Kiyohara, H., Cyong, J. -C. and Yamada, H. (1988) Carbohydr. Res., 182, 259-275.
Zhao, J. -F., Kiyohara, H. and Yamada, H. (1991) Carbohydr. Res., 219, 149-172.
Renard, C. M. G. C. and Thibault, J. -F. (1993) Carbohydr. Res., 244, 99-114.

Rees, D. A. and Richardson, N. G. (1966) Biochemistry, 5, 3099-3107.

Wood, P. J. and Siddiqui, I. R. (1972) Carbohydr. Res., 22, 212-220.

Aspinall, G. O. and Cottrell, I. W. (1971) Can. J. Chem, 49, 1019-1022.

Neukom, H., Deuel, H, Heri, W. J. and Kundig, W. (1960) Helv. Chim. Acta, 43,
64-71.

Siddiqui, I. R. and Wood, P. J. (1974) Carbohydr. Res., 36, 35-44.

Knee, M. (1970) J. Exp. Botany, 21, No. 68, 651-662.

Aspinall, G. O. and Jiang, K. -S. (1974) Carbohydr. Res., 38, 247-255.

York, W. S., Darvill, A. G., McNeil, M., Stevenson, T. T. and Albersheim, P. (1985)
Methods Enzymol., 118, 3-40.

Thibault, J. -F. (1983) Phytochemistry, 22, 1567-1571.

94.

95.

96.

97.

98.

99.

29
Joseleau, J. -P., Chambat, G. and Lanvers, M. (1983), Carbohydr. Res., 122, 107-
113.
Cheng, L. and Kindel, P. K. (1995) Analy. Biochem., 288, 109-114.
Wood, P. J. and Siddiqui, I. R. (1971) Anal. Biochem., 39, 418-428.
Aspinall, G. O. and Fanous, H. K. (1984) Carbohydr. Polymers, 4, 193-214.
Saulnier, L. and Thibault, J. -F. (1984) Carbohydr. Polymers, 7, 329-343.

Massiot, P., Rouau, X. and Thibault, J. -F. (1988) Carbohydr. Res., 172, 229-242.

100. Marcelin, 0., Saulnier, L. and Brillouet, J. -M. (1991) Carbohydr. Res., 212, 159-

167.

101. Renard, C. M. G. C., Voragen, A. G. J., Thibault, J. -F. and Pilnik, W. (1990)

Carbohydr. Polymers, 12, 9-25.

CHAPTER II

ISOLATION OF CELL WALLS AND SOLUBILIZATION OF PECTIC

POLYSACCHARIDES FROM Lemna minor

[In part from Kindel, P. K., Cheng, L., and Ade, B. R. (1996) Phytochemistry, 41, 719-723.]

30

31

ABSTRACT

Improved procedures for isolation and purification of cell walls of L. minor and
extraction of pectic polysaccharides from the cell walls were developed. Plants were
homogenized with a Waring blender and the homogenate was treated with a French press.
This treatment resulted in a much more complete breakage of cell walls and less
contamination by other cell components, such as chloroplasts, than cell walls homogenized
only with a Waring blender. Fifty-six percent (56%) more pectic polysaccharide was
extracted by ammonium oxalate from the cell walls prepared with both a Waring blender and
a French press than from those prepared only with a Waring blender. Pectic polysaccharides
extracted from the cell walls that were prepared by homogenizing plants with salt (1 M
NaCl) or detergent (1% sodium deoxycholate) solution were similar in molecular size and
protein content. Pectic polysaccharides, which accounted for 26.4-31.7% of the total
anhydrogalacturonic acid in cell walls of L. minor, were solubilized with 0.05 M ammonium
oxalate at 22°C in 30 min. Further extracting the cell wall residues with 0.05 M ammonium
oxalate for 6 h solubilized only 6.4% more pectic polysaccharides. Extraction of the cell
wall residue, following ammonium oxalate extraction, with 0.05 M NaQCO3/0.02 M NaBH4
at 4°C for 17 h followed by 0.05 M NaQCO3 at 23°C for 3 h resulted in solubilization of less
than 0.5% of the total anhydrogalacturonic acid of the cell walls. A fast and effective method

of cell wall isolation was established.

32

INTRODUCTION

Methods for cell wall preparation and polysaccharide isolation vary with different
plants and cell types. Methods considered to be generally suitable for isolation of pectic
polysaccharides from parenchymatous tissue of vegetables and fruits, lignified tissues, and
cereals as well as special techniques used for starch- and protein-rich tissues have been
described (1). The quantity of extractable material varies with different methods, type of
plant tissue and the stage of development of the cell wall but it has not been possible to
completely extract all pectic polysaccharides from any plant material by any method
without causing degradation of the polysaccharides (1,2). However, effort should be made
to minimize degradation caused by enzyme activity and to avoid contamination by
intracellular compounds, such as starch and cytoplasmic proteins, when cell walls are
prepared.

A number of different methods have been used to break cell walls of parenchymatous
tissues, such as homogenizing with a Waring blender (3), ball-milling (4), grinding with
a pestle and mortar followed by blending with a Ultra-Turrax (5), vibrating with a cell mill
containing glass beads (6,7) and using a French pressure cell (8-10). A Parr bomb (11),
and a French pressure cell plus sonication (12) were used to break suspension cultured
single plant cells successfully. When pectic polysaccharides were isolated from L. minor
a number of years ago (3) the cell walls were prepared by homogenizing the plants with
a Waring blender. The purity of the cell walls was not established. Re-examination of such

preparations microscopically showed that disruption of cells was incomplete (see Results).

33

In this study, the cell walls were prepared by homogenizing the plants with a Waring blender
followed by treatment with a French press. The effectiveness of using a French press was
evaluated.

Homogenization of cell walls in solutions of SDS and SDC is considered to be an
effective way of removing cytoplasmic proteins (4,5). SDC (0.5 and 1%w/v) is preferred
by some investigators because homogenization in SDS results in excessive foaming (13-16).
Since SDS interferes with color development in the uronic acid assay (17) and SDC
solubilizes the glycoprotein cell walls of an alga ( 18), the necessity of using detergent in the
homogenization of L. minor was examined in this work.

Recent research on the kinetics of solubilization of pectic polysaccharides from cell
walls of L. minor showed that ~30% of the total AUA of the cell walls was solubilized by
ammonium oxalate in 15 min and that extending the time of extraction to 5 h only resulted
in 4% more total AUA being solubilized (l7). Jarvis et al. (16) reported that 0.1 M Na2C03
was able to extract the remaining galactan-rich pectic polysaccharide effectively after the cell
walls of potato were extracted with chelating agents. After extraction with CDTA, extraction
with 0.05 M NaQCO3 solubilized an additional 12.6% and 8.1%, respectively, of the pectic
polysaccharides from the cell wall residues of onions and apples (4). Selvendran et al. (4)
proposed a general procedure for sequential extraction of cell wall polysaccharides from
parenchymatous tissues which involved the use of 0.05 M NaQCO3/0.02 M NaBI-L at 1 °C and
0.05 M NaQCO3 at 22°C after the chelator extraction. The effectiveness of this procedure

with L. minor was studied in this work.

34

MATERIALS AND METHODS

Materials and general methods. DEAE-Sephadex A—25 [particle (bead) size: 40-120
pm, dry; 80-242 um, wet], Sephacryl S-400, and MF—Millipore filters were purchased
from Pharmacia Biotech, Inc., Sigma Chemical Co., and Millipore Corp. , respectively.
BCA reagent was obtain from Pierce. Total protein in purified cell walls was determined
with BCA reagent and BSA was used as the protein standard (19). Uronic acid and total
sugar were determined with 3-hydroxydiphenyl (20) and phenol-sulfuric acid (21),
respectively. Total AUA in cell walls was determined (17). The determinations of uronic
acid and neutral sugar in filtrates were corrected for mutual interference as described (17).
In the preparation of cell walls and solubilization of pectic polysaccharides from the cell
walls, all flitrations were done with 15 um Nylon mesh (3-15/6, Tetko, Inc.).

Preparation of cell walls. L. minor was grown as described elsewhere (22). The
cell walls were prepared from whole plants of L. minor at 4°C, basically with the
procedure of Kindel et al. (Appendix A and Ref. 17) except for the following changes:
Plants (245.3 g, wet wt) were homogenized with 600 ml of 1.0 M NaCl in a Waring
blender. The homogenate was filtered, and the cell walls resuspended in 500 ml of water
and filtered. Two weighed portions of cell walls, each 0.0632 i 0.0043 g, wet wt, were
removed from the residue (97 .9 g, wet wt) and dried to constant weight in vacuo and over
P205. The rest was divided into two equal weight parts: one part was called Cell Wall-W
(cell walls prepared by homogenizing with a Waring blender only) and saved for extraction

of pectic polysaccharides. The other part was suspended in 300 mL water, passed through

35

a French pressure cell (at 16000-18000 psi), filtered, resuspended in 250 mL of water,
filtered and the residue was called Cell Wall-F. Two weighed portions of Cell Wall-F
(34.05 g, wet wt), each 0.042 i 0.002 g, wet wt, were dried as described above and the
rest was saved for extraction of pectic polysaccharides. In a separate experiment, cell
walls were prepared from 70 and 50 g of plants as described for the preparation of Cell
Wall-F but the homogenization was conducted in 170 ml of 1% (w/v) SDC and 120 mL
of 1 M NaCl, respectively, and the cell walls were called Cell Wall-SDC and Cell Wall-
NaCl, respectively. The solutions of the cell wall preparations following filtration were
combined and analyzed for uronic acid and total sugar. Total protein was determined for
Cell Wall-SDC and Cell Wall-NaCl.

Solubilization of pectic polysaccharides from cell walls. Cell Wall-W and Cell
Wall-F were extracted with 585 mL and 500 mL, respectively, of 0.05 M ammonium
oxalate (pH 6.0) at 22°C for 20 min, filtered, resuspended in 100 mL water and filtered.
The residues and combined filtrates were tested for uronic acid. In a separate experiment,
31.9 g of fresh cell walls that were prepared by the same procedure used to prepare Cell
Wall-F were extracted with 530 mL of 0.05 M ammonium oxalate for 30 min, filtered,
resuspended in 300 mL water and filtered. The ammonium oxalate extraction of the
residue was repeated once, this time for 6 b. Two further extractions of the ammonium
oxalate residue were conducted with 180 mL of 0.05 M NazCO3/0.02 M N aBH4 at 4°C for
17 h, and with 0.05 M NaQCO3 at 23°C for 3 h, respectively. Cell wall residues and filtrates
were tested for uronic acid.

. Cell Wall-SDC and Cell Wall-NaCl were extracted with 190 mL and 150 mL, of 0.05

36

M ammonium oxalate at 22°C for 30 min and centrifuged. The supernatant solution was
collected, dialyzed against distilled water for 38.5 h and called P-SDC and P-NaCl,
respectively, for polysaccharides extracted from Cell Wall-SDC and Cell Wall-NaCl. P-
SDC (345 mL) and P-NaCl (262 mL) were tested for total sugar and uronic acid and the
remainder of each was concentrated and fractionated by chromatography with DEAE-
Sephadex.

In a large scale experiment, cell walls prepared from 597 g (wet wt) of plants (as the
preparation of Cell Wall-F) was extracted with 1070 mL 0.05 M ammonium oxalate (pH
5.5), filtered, resuspended in 850 mL of 0.05 M ammonium oxalate and filtered. The
filtered solutions were combined and tested for uronic acid. Purification and
characterization of this pectic polysaccharide fraction are described in Chapters V and VI
of this thesis.

Fractionation of P-SDC and P-NaCl on DEAE-Sephadex. Solutions of
polysaccharides P-SDC and P-NaCl were concentrated to 114 mL and 74 mL, respectively,
and filtered with a 5 pm membrane (MP-Millipore, Millipore Corp.). The filtered samples
were adjusted to pH 7.7 and 0.067 M sodium phosphate with 0.67 M sodium phosphate
buffer (pH 7.6), and each was applied to a column of DEAE-Sephadex [1.3 cm (i.d.) x 13.9
cm (h), and 1.05 cm (i.d.) x 12.4 cm (h), respectively]. The columns were developed with
580 and 340 mL, respectively, of 0.067 M sodium phosphate buffer, pH 7.7, containing a
linear gradient of NaCl from 0 to 0.4 M. Fractions of 4.3 and 2.5 mL were collected at rate
of 0.21 and 0.13 mL/min, respectively. Each fraction were tested for total sugar and uronic

acid. Appropriate fractions were combined as P-SDC-I, P-SDC-II, P-NaCl-I and P-NaCl-II

37
(these are defined in Figure 2.2). They were dialyzed, concentrated and analyzed by size
exclusion column chromatography.

Size exclusion column chromatography. P-SDC-I, P-SDC-II, P-NaCl-I and P-NaCl-II
were concentrated at 35°C to 1-3 mL and applied to a Sephacryl S-400 column (1.05 cm, id
x 83 cm, h). The column was developed with 0.1 M NaC1/0.05 M sodium phosphate (pH
6.5) at a flow rate of 0.1] mL/min. Fractions of 1.5 mL were collected and analyzed for
uronic acid. The relative molecular size of each polysaccharide was determined by

comparing their mobilities with those of Dextran T-500, T-70 and T-10.

RESULTS

Comparison of cell walls disrupted with the Waring blender and French press. The
color of Cell Wall-W was green. Under 330x magnification, intact cells (with chloroplasts)
and multicellular pieces of tissue were observed (Figure 2.1a). Cell Wall-F was cream-
colored and appeared to be free of chloroplast and other cell organelles. Few intact cells
remained in Cell Wall-F (Figure 2.1b). The pectic polysaccharides solubilized (determined
as AUA) from Cell Wall-W and Cell Wall-F with 0.05 M ammonium oxalate in 20 min were
13.1% and 20.4% (w/w), respectively, of the total AUA found in cell walls, which means
that treatment of the cell walls with both the Waring blender and the French resulted in a
56% increase of pectic polysaccharides released from the cell wall.

Media used for cell wall homogenization. In the 30 min extraction of Cell Wall-SDC

and Cell Wall-NaCl (after dialysis) with 0.05 M ammonium oxalate, pectic polysaccharides

38

Figure 2.1. Light microsc0pic view of cell walls of L. minor. (a) Cell Wall-W (330x) , the
cell walls prepared by homogenizing the plant with a Waring blender. (b) Cell Wall-F
(530x), the cell walls prepared by homogenizing the plants with a Waring blender plus a

French press.

39

g "It,”
.1, ” Dismptecl.
.cell wall

 

40

that are 24.4% and 22.6%, respectively, of the total AUA in the cell walls were solubilized
while pectic substances (including mono-, oligo- and polysaccharides) present in the filtrates
obtained during preparation of cell walls were 5.7% and 10.1%, respectively, of the total
AUA in the cell walls (Table 2.1). On the dry weight basis, Cell Wall-SDC and Cell Wall-
NaCl contained nearly the same amount of protein (Table 2.1).

The elution profiles from DEAE-Sephadex column chromatography of P-SDC and P-
NaCl are shown in Figure 2.2. Two polysaccharide fractions were obtained from P-SDC,
named P-SDC-I and P-SDC-II (Figure 2.2a), and two from P-NaCl, named P-NaCl-I and
P-NaCl-II (Figure 2.2b). Fractions under each peak were combined as indicated (Figure 2.2).
Size exclusion chromatography showed that the relative molecular weights of
polysaccharides P-SDC-I, P-NaCl-I, P-SDC-II, and P—NaCl-I are 1 1,600, 12,000, 18,700 and
18,200, respectively (Table 2.1).

Sequential extraction of pectic polysaccharides from cell walls. The total AUA found
in cell walls of L minor (Cell Wall-F) is 18.7% (w/w) on a dry weight basis (Table 2.2).
The pectic polysaccharides solubilized from Cell Wall-F and the cell walls of large scale
experiment by 0.05 M ammonium oxalate in 30 min were 26.4% and 31.7%, respectively,
of total AUA in the cell walls, which were acceptably close. Further extraction of the residue
with ammonium oxalate for up to 6 h only resulted in 6.4% more pectic substance being
solubilized (Table 2.2). The extraction of additional pectic substances from the cell wall
residue of the ammonium oxalate extraction with weak base is difficult; only 0.20% and
0.24% more pectic substance were solubilized with the sequential treatments of 0.05 M

NaQCO3/0.02 M NaBH4 at 4°C for 17 h followed by 0.05 M at 23°C for 3 h (Table 2.2).

41

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42

Figure 2.2. Column chromatograms of L. minor cell wall pectic polysaccharide
fractions P-SDC (a) and P-NaCl (b). Columns were developed with 0.067 M sodium
phosphate buffer, pH 7.7, and buffer containing a linear gradient of NaCl ( ----- - ). Uronic

acid (A520 m, -o— ) and total sugar (A490 "m, ------ 0 ------ ) were determined as described in

the Materials and Methods.

Absorbance

Absorbance

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45

DISCUSSION

Homogenizing L. minor with a Waring blender (3) resulted incomplete disruption of
cells since many intact cells with chloroplasts and multicellular pieces of tissue were
observed in Cell Wall-W. However, after homogenization with a Waring blender, the cell
wall suspension can be processed readily with a French pressure cell and this resulted in
almost complete disruption of cells. It is not surprising to find that 56% more pectic
polysaccharides were extracted by ammonium oxalate form Cell Wall-F than from Cell
Wall-W. With the more thoroughly disrupted cell walls, the chelating reagent has a
greater chance to interact with cell wall Ca“ ions and release pectic polysaccharides held
by Ca2+ bridges. When the French pressure cell was used the cell walls (Cell Wall-F) were
cream-white and appeared to be free of chloroplast and other cell organelles under
microscope while Cell Wall-W was green.

Cell walls isolated by homogenization with l M NaCl or 1% SDC were basically the
same since: (i) the protein content of each type of cell wall is basically the same, (ii) the
percentage of pectic substances solubilized with 0.05 M ammonium oxalate in 30 min from
Cell Wall-NaCl, 22.6%, was fairly close to that solubilized from Cell Wall-SDC, 24.4%, (iii)
no degradation of the polysaccharides resulted from using 1% SDC for 1 M NaCl as the
homogenization media, based on the finding that the relative molecular sizes of the two
pectic polysaccharides isolated from Cell Wall-SDC and Cell Wall-NaCl are basically the
same (Table 2.1). Detergent, such as SDS, was interferes with color formation in the uronic

acid test (17). Some foaming was observed when using 1% SDC as the homogenization

46

medium. Consequently N aCl solution is preferred as the homogenization medium.
Although the amount of pectic polysaccharides solubilized from Cell Wall-F (26.4%)
with 0.05 M ammonium oxalate in 30 min was somewhat lower than that of the large scale
experiment (31.7%) and that reported (30%) by Kindel et al. (17), it is clear that the release
of ammonium oxalate soluble pectic polysaccharides from L. minor is not a continuum and
most of these molecules were solubilized within 30 min with 0.05 M ammonium oxalate.
The determination of the kinetics of solubilization showed this conclusively (17). Further
extraction of the cell wall residue for 6 h (these results) and 5 h (17) resulted in 6.4% and
4%, respectively, more pectic polysaccharides being extracted. The pectic substances in the
cell wall residue from ammonium oxalate extraction were resistant to extraction with weak
base —only 0.4% of the total AUA in the cell walls was solubilized by the two weak base
extractions. If the remaining AUA represents pectic polysaccharides of the cell walls, these
pectic polysaccharides should be different than the chelator soluble ones, at least their
bonging in the cell wall does not depend on Ca2+ bridge since they are not solubilized by the
chelator. Also their attachment to the cell wall appears not to involve an ester linkage, since
they are resist to weak base extraction. Since the solubilization process is fast (no significant
additional release of AUA after 30 min) and the conditions of solubilization are non-
destructive, the breakage of acid or base labile glycosidic linkages under extraction

conditions should be minimal.

10.

11.

12.

47

REFERENCES

Selvendran, R. R. and O'Neill, M. A., (1987) Methods Biochemical Analysis 32,
25-153.

Selvendran, R. R. (1985) J. Cell Sci. Suppl. 2, 51-88.

Hart, D. A. and Kindel, P. K. (1970) Biochem. J. 116, 569-579.

Selvendran, R. R., Stevens, B. J. H. and O’Neill, M. A. (1985) Biochemistry of
Plant Cell Walls, Society for Experimental Biology, Seminar seriese: 28,
Cambridge University Press, 39-78.

Selvendran, R. R. (1975) Phytochemistry, 14, 1011-1017.

Northcote, D. H., Goulding, K. J. and Home, R. W. (1958) Biochem. J., 70, 391-
397.

Lamport, D. T. A. (1965) in Advances in Botanical Research, (R. D. Preston, Ed.)
Vol 2, pp. 151-218, Academic Press, New York and London.

Bacon, S.D., Gordon, A. H. and Morris, E. J. (1975) Biochem. J., 149, 485-487.
Nakamura, N. and Suzuki, H. (1981) Phytochemistry, 20, 981-984.

Takeda, H. and Hirokawa, T. (1978) Plant Cell Physiol., 19, 591-598.

York, W. S., Darvill, A. G., McNeil, M., Stevenson, T. T. and Albersheim, P.
(1986) Methods Enzymology, 118, 3-40.

Harris, P. J . (1983) in Isolation of Membranes and Organelles from Plant Cells,
(Hall, J. L. and Moore, A. L., Bd.), pp. 25—53, Academic Press, New York and

London.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

48

Klis, F. M. and Eeltink, H. (1979) Planta, 144, 479-484.

Mankarios, A. T., Hall, M. A., Jarvis, M. C., Threlfall, D. R. and Friend, I.
(1980) Phytochemistry, 19, 1731-1733.

Van Host, G. J., Klis, F. M., Bouman, F. and Stegwee, D. (1980) Planta, 149,
209-212.

Jarvis, M. C., Hall, M. A., Threlfall, D. R. and Friend, J. (1981) Planta, 152, 93-

100.

Kindel, P. K., Cheng, L. and Ade, B. R. (1995)Phytochemistry, Vol. 41, No. 3,
719-723.

Roberts, K. (1974) Philos. Trans. R. Soc. London, Ser B., 268, 129-146.
Redinbaugh, M. G. and Turley, R. B. (1986) Anal. Biochem., 153, 267—271.
Blumenkrantz, N. and Asboe-Hansen, G. (1973) Anal. Biochem. 54, 484-489.
DuBois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A. and Smith, F. (1956)
Anal. Chem. 28, 350-356.

Kindel, P. K. and Watson, R. R. (1973) Biochem. J. 133, 227-241.

CHAPTER III

EFFECT OF CATIONS ON THE ELUTION OF PECTIC POLYSACCHARIDES

FROM ANION-EXCHANGE RESINS

[Adapted from Cheng, L. and Kindel, P. K. (1995) Analy. Biochem., 228, 109-114]

49

50

ABSTRACT

Conditions for quantitatively eluting six plant pectic polysaccharides from
diethylaminoethyl(DEAE)-columns were established. Surprisingly, the cation greatly
affected the elution of pectic polysaccharides from these anion exchange columns. Elution
of apple pectic acid from DEAE-Sephadex A-25 and DEAE-Trisacryl Plus-M columns was
quantitative with 0.5 M of NH4CI, LiCl, or CsCl in 0.05 M acetate buffer (pH 5.5). In
contrast, sample recovery from the two columns was only 6.3% and 8.6%, respectively, with
up to 1 M NaCl in buffer, and 54% and 32%, respectively, with l M KCl in buffer. In each
case, the retained apple pectic acid was eluted quantitatively by 0.5 M NH4C1 in buffer. The
elution of four plant pectic polysaccharides —one from Apium graveolens (celery), two from
L. minor (duckweed) and a commercial citrus polygalacturonic acid —-from columns of
DEAE-Trisacryl was incomplete when the columns were developed with 0.5 M NaCl or 0.5
M KCl in buffer; the recovery of the samples from the columns ranged from 0 to 89%.
However, again, the retained portion of each sample was eluted quantitatively or almost so
from the columns with 0.5 M NH4C1 in buffer. Elution of a commercial citrus pectin from
DEAE-Trisacryl columns was accomplished equally well and almost quantitatively by 0.5
M N aCl, KCl, and NH4C1 in buffer. On a preparative DEAE-Trisacryl Plus M column, 99%
of the pectic polysaccharides extracted from cell walls of L. minor was eluted by a gradient
of 0 to 0.5 M NH4C1 in buffer, while, in a basically identical experiment only 55% of the
sample was eluted when NH4C1 was replaced by NaCl in buffer. The discovery that NH;

ion in the eluent brings about the essentially quantitative elution of pectic polysaccharides

51

from anion exchange resins solves the long-standing problem of incomplete recovery of
pectic polysaccharides from DEAE-columns. Li+ and Cs+ ions are similar to NH," ion in

elution effectiveness.

INTRODUCTION

The separation and purification of pectic polysaccharides solubilized from plant cell
walls typically involves anion exchange column chromatography. A common problem in the
purification of cell wall polysaccharides is that the elution of the polysaccharide from the
anion exchange column is incomplete. Since (i) pectic polysaccharides extracted from cell
walls are usually both chemically and physically heterogeneous, (ii) polysaccharide matreials
retarded in the column can be structurally different from the eluted ones, analyzing the
sample that is incompletely recovered from the column can result in biased structural
information. Incomplete elution has been observed with a variety of pectic polysaccharides
from a number of different plants with all the different ion exchange resins commonly used
for purification of pectic polysaccharides. When DEAE-cellulose (1,2), DEAE-Sephadex
(both A-25 and A-50) (3-6), DEAE-Sephacel (7-12) and DEAE-Sepharose CL-6B (13,14)
were used, the recoveries of pectic polysaccharides from columns where elution was
incomplete ranged from 82 to 85%, 0 to 87%, 32 to 88%, and 24 to 51%, respectively. When
DEAE-Trisacryl M was used, the recoveries of pectic polysaccharides ranged from 69 to
88% (15,16). Recoveries of 94 and 100% were obtained with the soluble portion of two

freeze-dried pectic polysaccharide fractions of runner bean pods when DEAE-Trisacryl was

52

used (17); however, the portion of the freeze-dried fraction that was soluble was only 85 and
27%, respectively, of the total fraction. Redgwell and Selvendran compared the recoveries
of an onion pectic polysaccharide fraction from four different anion exchange resins at either
pH 4.5 or 6.3 or both (15). The recoveries from DEAE-Sephadex, DEAE-Sephacel, and
DEAE-Sepharose ranged from 44 to 60%. The recovery was 88% when DEAE-Trisacryl
was used and 0.05 M phosphate (pH 6.3) and 0.5 M NaCl in buffer were the eluents, but this
dropped to 51% when DEAE-Trisacryl, 0.05 M acetate buffer (pH 4.5), and 0.5 M NaCl
were used. When a pectic polysaccharide fraction from purified cell walls of L. minor was
applied to columns of DEAE-Sephadex A-25 and DEAE-Trisacryl Plus-M and the sodium
form of the eluents was used, the recovery of pectic polysaccharides ranged between 48 and
57% (Results).

We have found that the type of cation present in the eluent profoundly affects the elution

of pectic polysaccharides from anion exchange resins.

MATERIALS AND METHODS

Materials and general methods. DEAE-Sephadex A-25 [particle (bead) size: 40-120
um, dry; 80—242 um, wet], DEAE-Trisacryl Plus-M (bead size: 40-80 pm, wet), and MF-
Millipore filters were purchased from Pharmacia Biotech, Inc., Sigma Chemical Co., and
Millipore Corp., respectively. Uronic acid was determined colorimetrically (18) and is
expressed as AUA (molecular weight: 176.12). Apple pectic acid was purchased from

Aldrich Chemical Co. Polygalacturonic acid and citrus pectin were obtained from Sigma

53

Chemical Co. The suppliers provided the following information: (i) apple pectic acid —— 1V1w
228,000; M“, 88,000; AUA content, approximately 75%; degree of methyl esterification, less
than 5%, (ii) citrus polygalacturonic acid — approximate molecular weight range, 4000 to
6000; AUA content, approximately 100%; degree of methyl esterification, approximately
5%, and (iii) citrus pectin — approximate molecular weight range, 23,000 to 71,000; AUA
content, approximately 76%; degree of methyl esterification, approximately 50%. Pectic
polysaccharides of L. minor were prepared from highly purified cell walls. Plants were
grown as described previously (19). Cell walls of L. minor were prepared as described
(Appendix A, Ref. 20) and were extracted with 0.05 M ammonium oxalate (pH 5.5) at 22°C
for 30 min. The pectic polysaccharides in the extract were chromatographed on DEAE-
Trisacyl and column fractions were tested for uronic acid. Two pectic polysaccharides were
isolated; polysaccharide F-129, which consisted of 54% neutral sugars and 46% galacturonic
acid and had a relative molecular weight (relative to standard dextrans) of 480,000, and
polysaccharide F—167, which consisted of 7% neutral sugars and 93% galacturonic acid and
had a relative molecular weight of 780,000 (Cheng and Kindel, 1996, submitted). A pectic
polysaccharide fraction from Apium graveolens (celery) was prepared by extracting purified
cell walls of celery with 0.05 M ammonium oxalate (pH 5.5) at 22°C for 1 h (20).
Analytical anion exchange column chromatography of pectic polysaccharides. Apple
pectic acid, polygalacturonic acid, and citrus pectin were dissolved by stirring each in water
and adding 5% (w/v) NaOH to bring the pH to 5.5. The solutions were filtered through a 0.8
pm MF—Millipore filter. Polysaccharide concentrations up to 2 mg/mL were prepared.

Apple pectic acid (6 mg in 3 mL of water at pH 5.5) was applied to columns of DEAE-

54

Sephadex A-25 and DEAE-Trisacryl Plus M. The columns [0.7 cm (diameter) x 8.5 cm
(height)] were developed with 20 mL of column buffer [0.05 M sodium acetate, potassium
acetate, ammonium acetate, lithium acetate, or cesium acetate (pH 5.5)], 40 mL of 0.5 M salt
(NaCl, KCl, NH4C1, LiCl, or CsCl) in 0.05 M of the corresponding column buffer, and 25
mL of 1 M salt (NaCl, KCl, NH4C1, LiCl, or CsCl) in column buffer. The columns
developed with NaCl or KC] in column buffer were further developed with 40 mL of 0.5 M
NH4C1 and 20 to 25 mL of 1 M Nle each in 0.05 M ammonium acetate (pH 5.5). The
columns developed with LiCl or CsCl in column buffer were further developed with 20 to
25 mL of 1 M NH4C1 in 0.05 M ammonium acetate (pH 5.5).

A celery pectic polysaccharide fraction (approximately 0.8 mg AUA in 20 mL of 0.05 M
ammonium oxalate at pH 5.5), citrus polygalacturonic acid (6 mg in 6 mL of water at pH
5.5), citrus pectin (6 mg in 6 mL of water at pH 5.5), pectic polysaccharide F-129 from L.
minor (0.93 mg AGalU in 6 mL of water), and pectic polysaccharide F-167 from L minor
(1.1 mg AUA in 3 mL of water) were applied to individual DEAE-Trisacryl Plus M columns
and eluted with the salts stated in Table 3.2. For citrus polygalacturonic acid and citrus
pectin, the columns were the same size as above and developed as described above. The
final treatment for these six columns was with 0.5 M NH4C1 in water at pH 9.5 (adjusted
with NI-LOH; final [NH,*] was 0.64 M). For the celery pectic polysaccharide fraction and
polysaccharides F-129 and F-167, the columns were 0.59 cm (diameter) x 6 cm (height). The
columns were also developed basically as described above through treatment with 0.5 M
NH4C1 in buffer, with a proportional reduction in solution volumes. With these three

samples, all salts were dissolved in 0.05 M sodium acetate (pH 5.5). Polysaccharide F-167

55

was also directly eluted from a DEAE-Trisacryl Plus M column with 0.5 M LiCl in buffer.
Column eluates were tested for AUA colorimetrically (18). The yield of pectic
polysaccharide from the columns was based on recovery of AUA in the eluates.
Preparative anion exchange column chromatography of a pectic polysaccharide fraction
from L.m_in_gj. Cell walls and ammonium oxalate extracts of L. minor were prepared as
described above. The extracted pectic polysaccharides (178 mg of AUA equivalents) in 0.03
M sodium oxalate (pH 5.0) (column buffer) were applied to a column of DEAE-Sephadex,
1.75 cm (diameter) x 18 cm (height). The column was washed with 0.05 M sodium acetate
(pH 5.0, column buffer) and developed sequentially with 1600 mL of column buffer
containing a linear gradient of NaCl that increased from 0 to 0.4 M, 168 mL of 1M NaCl in
column buffer, 240 mL of 1 M NaCl at pH 2.0 and 156 mL of 1 M NaCl at pH 1.2.

In another large scale experiment, the extracted pectic polysaccharides, 192 mg (AUA
equivalents), in 0.05 M sodium oxalate buffer (pH 5 .0, column buffer) were fractionationed
with a DEAE-Trisacryl column (2.8 cm, id, x 28 cm, height). The column was sequentially
developed with 2550 mL of column buffer containing a linear gradient of 0 to 0.5 M NaCl,
191 mL of 1 M N aCl in column buffer and 229 mL of 1 M NaCl in 0.05 M sodium
phosphate (pH 8.2). In an experiment with an identical column, 210 mg (AUA equivalents)
of extracted pectic polysaccharides from cell walls of L. minor were fractionated with 2800
mL of ammonium oxalate (pH 5.5, column buffer) and a linear gradient of 0 to 0.5 M NH4C1
followed by 520 mL of 1M ammonium oxalate in the column buffer. Column fractions were
assayed for uronic acid.

Effect of salts on the solubility of apple pectic acid. Solutions in test tubes, 1.03 cm

56
(i.d.) x 7.5 cm (length), contained: (1) apple pectic acid at 0.5 or 1.0 mg/mL and (ii) NaCl,
KCl, NH4Cl, LiCl, or CsCl at 0.5, 1.0, 1.5 or 2 M in a final volume of 4.0 mL. The solutions
were prepared from stock solutions and after mixing stood at 22°C. Precipitation and gel
formation were detected by measuring absorbance at 600 nm. Measurements were taken at

0.5, 6, 16, 28, 52, 75 and 102 h after mixing.

RESULTS

The effect of cations on the recovery of apple pectic acid from DEAE-Sephadex and
DEAE-Trisacryl columns is summarized in Table 3.1. When DEAE-Sephadex and DEAE-
Trisacryl columns were developed with 0.5 M NH4C1 in column buffer, apple pectic acid was
eluted immediately and quantitatively (97%, Table 3.1). When the columns were developed
with 0.5 M LiCl or CsCl in column buffer, the recovery of apple pectic acid was almost
quantitative, ranging from 88 to 96% (Table 3.1). The remainder was recovered by eluting
with the corresponding 1 M salts and 1 M NH4C1 in buffer. In contrast, when 0.5 M NaCl
in column buffer was the eluent, the recovery of apple pectic acid from DEAE-Sephadex and
DEAE-Trisacryl columns was 8.6 and 6.3%, respectively (Table 3.1). When 0.5 M KCl in
column buffer was used for column development, somewhat better recoveries were obtained
from both DEAE-Sephadex and DEAE-Trisacryl (54 and 32%, respectively). Further
development of the columns with 1 M NaCl or KCI in column buffer did not result in the
elution of any additional pectic acid. However, development of the columns with 0.5 M

NH4C1 in buffer eluted the remaining apple pectic acid from both columns basically

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58

quantitatively.

When apple pectic acid was applied to DEAE-Trisacryl columns and the columns were
developed with either 0.5 M NaCl or 0.5 M KCl in column buffer, a liquid-filled gap of
approximately 1 cm developed in the column bed between a top layer of approximately 1.5
cm and the rest of the column bed. The top layer did not shrink or collapse during the elution
with 0.5 M and 1 M NaCl or KC]. When the columns were treated with 0.5 M NH4C1 in
column buffer, the top layer collapsed. The collapse corresponded with the quantitative
elution of the remainder of the apple pectic acid from the columns. When DEAE-Trisacryl
columns were developed only with 0.5 M NH4Cl, 0.5 M LiCl or 0.5 M CsCl in column
buffer, there was no separation of the resin bed as the resin shrunk. Also no gap in the resin
bed developed when DEAE-Sephadex was used in any of the above experiments including
those with NaCl and KC].

The effect of cations on the recovery of five different pectic polysaccharide samples
from DEAE-Trisacryl columns is summarized in Table 3.2. The recoveries of four were
incomplete, ranging from 0 to 89%, when elution was with 0.5 M NaCl or KC] in buffer.
With one polysaccharide, citrus pectin, elution was near quantitative and it made no
difference whether N a“, K”, or NH,+ ion was present in the eluent. The retained portion of
the celery pectic polysaccharide fraction and of pectic polysaccharide F—129 from L. minor
following column chromatography with NaCl and KC] was eluted quantitatively with 0.5 M
NH4C1 in buffer. Pectic polysaccharide F- 167 from L. minor, which was completely retained
when NaCl or KCl was the eluent, was also eluted by 0.5 M NH4C1 in buffer, almost

quantitatively. Most, but not all, of the retained portion of citrus polygalacturonic acid was

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eluted by 0.5 M NH4Cl in buffer. Although only a small portion of citrus pectin was
retained, the retained material was also not eluted by 0.5 M NH4Cl in buffer. The retained
portion of citrus polygalacturonic acid and of citrus pectin following chromatography with
0.5 M NH4C1 was completely eluted by 0.5 M NH $1 in water at pH 9.5 (Table 3.2,
footnote). An experiment was performed to determine the pH required to elute the retained
portion of citrus polygalacturonic acid. Citrus polygalacturonic acid was applied to three
analytical columns of DEAE-Trisacryl. The columns were developed in order with 0.5 M
(83 to 86% of the AUA applied was eluted) and 1 M (2% was eluted) NH4Cl in buffer at pH
5.5. Treatment of the columns with 0.5 M NH4Cl in water at pH 6.5, 7.5, or 8.5 (pH adjusted
with NH4OH) eluted 0.4, 0.7, and 7.1% additional sample. The remainder was eluted from
the columns with 0.5 M NH4C1 in water at pH 9.5. The total recovery of sample from the
columns ranged from 96 to 99%. Pectic polysaccharide F—167 was very effectively eluted
with 0.5 M and 1.0 M LiCl (Table 3.2).

Similar results were obtained on a preparative scale. Recovery of AUA was 47.4%
when the DEAE-Sephadex column was treated with O to 0.4 M N aCl in column buffer (Table
3.3). Additional washing of the column with 1 M NaCl in column buffer and at pH 2.0 and
1.2 eluted only an additional 1.5% AUA (Table 3.3). Similarly, when the pectic
polysaccharides were fractionationed on a DEAE-Trisacryl column and eluted with O to 0.5
M NaCl in column buffer, 55% of the sample was eluted and washing the column with 1 M
NaCl in pH 5.0 column buffer and pH 8.2 buffer only resulted in the recovery of an
additional 0.4% of the sample (Table 3.3). However, when a linear gradient of 0 to 0.5 M

NH4Cl in column buffer was used to develope an identical colunm, 99% of the pectic

61

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62

polysaccharide material applied was eluted (Table 3.3).

Gel formation was observed when apple pectic acid at concentrations of 0.5 and 1
mg/mL was incubated with four different concentrations of NaCl and KC] (Figures 3.1a and
b). For apple pectic acid samples that contained both 0.5 mg/mL (Figure 3.1a) and 1 mg/mL
(Figure 3.1b), the opacity of the gel, as measured by absorbance at 600 nm, did not change
significantly between 0.5 h and 102 h after NaCl or KC] was mixed with the pectic acid,
although the absorbance of the samples that contained 1 mg/mL apple pectic acid is 1.9-4.0
times as high as the absorbance of the samples that contained 0.5 mg/mL. For samples that
contained apple pectic acid at 1 mg/mL, the highest absorbance of the gel was at l M NaCl
(absorbance ranged from 0.115 to 0.121 over the 102 h), while for samples that contained
0.5 mg/mL apple pectic acid the highest absorbance was observed at 0.5 M NaCl (absorbance
ranged from 0.037 to 0.039). Lower gel formation was observed with samples that
contained KCl; for samples containing 0.5 and 1 mg/mL of apple pectic acid the highest
absorbance was at 0.5 M KCl, and it ranged from 0.015 to 0.017 (Figure 3.1a) and from
0.028 to 0.032 (Figure 3.1b), respectively, over the 102 h. No detectable gel or precipitate
was observed with apple pectic acid samples of 0.5 and 1.0 mg/mL when 0.5 M and 1 M
NH4C1 (A50.003) or 0.5, 1, 1.5 and 2 M LiCl (AS0.004) or CsCl (A50.005) was present.
However, a precipitate was observed in all samples containing 1.5 M and 2 M NH4C1 and,
in contrast to the gels obtained with NaCl and KCl, the precipitate increased with time
(Figures 3.1a and b). Samples containing 1 mg/mL of apple pectic acid and 2 M NH4Cl
produced a precipitate that was more opaque and formed more rapidly than those containing

0.5 mg/mL pectic acid and 1.5 M NH4Cl.

63

Figure 3.1. Precipitation of apple pectic acid with NILCI, NaCl, KCl, CsCl and LiCl.
Precepitation was measured as absorbance at 600 nm after 0.5, 1, 1.5 and 2 M salts were

mixed with 0.5 mg/mL (a) and 1 mg/mL (b) of apple pectic acid and solutions stood at

22°C for 0.5, 6, 16, 28, 52, 75 and 102 h.

 

65

The five salts were tested for their effect on color formation in the uronic acid test. The
uronic content of standard galacturonic acid samples containing 1 M NaCl, KC], NH4Cl,

LiCl, and CsCl was the same, within :I: 3%, as the control, which contained no salt.

DISCUSSION

The type of cation present in the eluent dramatically affected the recovery of pectic
polysaccharides from columns of DEAE-Sephadex A-25 and DEAE-Trisacryl Plus M.
Quantitative or near quantitative recovery of all pectic polysaccharides from DEAE-resins
was achieved when up to 1 M NH,+ ion was used in the eluent. Similar high recoveries were
achieved with Li+ or Cs+ ion. The recovery of apple pectic acid from the columns was lowest
when N a” ion was in the eluent and next lowest with K” ion. The discovery that the presence
of NH4*, Li", or Cs+ ion in the eluent brings about the essentially quantitative elution of pectic
polysaccharides from anion exchange resins solves the long-standing problem of incomplete
recovery of pectic polysaccharides from DEAE-columns.

We suggest that pectic polysaccharide samples retained on DEAE-columns when 0.5 M
NaCl or KC] is the eluent form a gel around the resin beads at the top of the column. This
is supported by the observation that both 0.5 M and 1.0 M NaCl and KC] caused formation
of a gel when mixed with solutions of apple pectic acid and by the positive correlation
between the formation of a thicker, heavier gel with NaCl than with KC] and the lower
recovery of three of the polysaccharide samples from columns developed with NaCl

compared to those developed with KC]. This is also supported by the observation that a gap

66

formed in DEAE-Trisacryl columns when apple pectic acid was applied and the columns
were developed with 0.5 M NaCl or KC]. Gel formation presumably occurred when
polysaccharide collecting at the top of the column during loading reached a sufficiently high
concentration. Increasing the concentration of N aCl or KC] or changing the anion (data not
presented) or the pH of the eluent did not dissolve the gel on the column. We suggest the gel
was dissolved when the cation in the eluent was changed to NH; ion. The collapse of the
upper, separated layer of DEAE-Trisacryl columns on the lower layer in experiments with
apple pectic acid and N aC] and KC] plus the immediate, basically quantitative elution of all
polysaccharides when the cation was changed from 0.5 M Na+ or K+ ion to 0.5 M NH4+ ion
both indicated the gel was dissolved by NH4+ ion. Presumably Li+ and Cs+ ions would also
be effective in dissolving the gel on the column since their elution effectiveness is similar
to that of NH,+ ion. The formation of the gel is also dependent upon structure of pectic
polysccharides. With citrus pectin, gel formation apparently does not occur and with pectic
polysaccharide F-129 of L minor little occurs as indicated by the near quantitative recoveries
of these polysaccharides from the columns when N aCl and KC] are the eluting salts. Citrus
pectin may not form a gel because of its high degree of methyl esterification and pectic
polysaccharide F-129 from L. minor may not because the neutral sugars in the polysaccharide
may be present as a large number of small side-chains distributed relatively equally along the
main chain. F-129 has a neutral sugar content of 54%.

When solutions of apple pectic acid were mixed with NaCl or KC], the material that
formed was considered a ge] because the suspensions were translucent and there was little

settling of the material for extended periods after formation. Although no detectable

67

precipitate or gel was formed when apple pectic acid and l M NH4C1 were mixed, a
precipitate was observed at higher concentrations of NH4Cl. In contrast to the results with
NaCl and KC], the precipitate with NH4C] increased with time and was more like a true
precipitate, that is, the suspensions were more opaque and the material settle soon after
formation. Both LiCl and CsCl up to 2 M did not cause any detectable gel formation or
precipitate with apple pectic acid at concentrations up to 1 mg of polysaccharide per mL.

The putative pectic polysaccharide gel that formed around beads of DEAE-Trisacryl
when NaCl and KCl were used was apparently firmer than that which formed around beads
of DEAE-Sephadex since in the experiments with apple pectic acid a gap did not form in the
DEAE-Sephadex resin bed as it did in the DEAE-Trisacryl bed even though DEAE-
Sephadex contracted more than DEAE-Trisacryl at the same concentration of NaCl or KC].
A firmer gel could also account for the lower recovery of apple pectic acid from DEAE-
Trisacryl (6.3 and 32%) compared to DEAE-Sephadex (8.6 and 54%). A firmer gel may
form because of the smaller wet bead size of DEAE-Trisacryl. However, Redgwell and
Selvendran observed that the recovery of an onion pectic polysaccharide fraction from
DEAE-Sephadex was lower (59%) than from DEAE-Trisacryl (88%) when phosphate buffer
at pH 6.3 and l M NaCl were used (15).

Quantitative recovery of six different pectic polysaccharides from DEAE-columns was
achieved when NH,+ ion was present in the eluent. In contrast, incomplete recovery, and in
one case no recovery, was obtained with five of the polysaccharides when N a+ or K“ ion was
present in the eluent. With one polysaccharide, citrus pectin, NH], Na+ and K+ ions were

equally effective in the elution of the polysaccharide. Li+ and Cs+ ions acted similar to NH;

68

ion. We postulated that a polysaccharide gel formed on the DEAE-column when Na“ or K”
ion was present in the eluent and the gel was responsible for the incomplete elution of

sample. We also proposed that NH,+ ion had the ability to dissolve the gel.

10.

ll.

12.

13.

14.

15.

16.

1‘7.

69

REFERENCES

Neukom, H., Deuel, H., Heri, W.J., and Kundig, W. (1960) Helv. Chim. Acta 43, 64-71.
Siddiqui, I. R. and Wood, PJ. (1974) Carbohydr. Res. 36, 35-44.

Aspinall, G. O. and J iang, K.-S. (1974) Carbohydr. Res. 38, 247-255.

Aspinall, G. 0., Fanous, H.K. and Sen, AK. (1983) in Unconventional Sources of
Dietary Fiber (ed. I. Furda) Am. Chem. Soc. Sym. Series 214, Am. Chem. Soc.,
Washington, D. C., pp 33-48.

Stevens, B. J .H. and Selvendran, R. R. (1984) Phytochem. 23, 107-115.

Kato, Y. and Nevins, D. J. (1989) Plant Physiol. 89, 792-797.

Stevens, B. J. H. and Selvendran, R. R. (1984) Carbohydr. Res. 128, 321-333.
Stevens, B. J. H. and Selvendran, R. R. (1984) Carbohydr. Res. 135, 155-166.
Aspinall, G. O. and Fanous, H. K. (1984) Carbohydr. Polymers. 4, 193-214.
Saulnier, L. and Thibault, J .-F. (1987) Carbohydr. Polymers 7, 329-343.

Massiot, P., Rouau, X., and Thibault, J .-F. (1988) Carbohydr. Res. 172, 229-242.
Marcelin, 0., Saulnier, L., and Brillouet, J.-M. (1991) Carbohydr. Res. 212, 159-167.
Rombouts, F. M. and Thibault, J .-F. (1986) Carbohydr. Res. 154, 177-187.

Renard, C. M. G. C., Voragen, A. G. J., Thibault, J. -F. and Pilnik, W. (1990)
Carbohydr. Polymers 12, 9-25.

Redgwell, R. J. and Selvendran, R. R. (1986) Carbohydr. Res. 157, 183-199.

Ryden, P. and Selvendran, R. R. (1990) Carbohydr. Res. 195, 257-272.

Ryden, P. and Selvendran, R. R. (1990) Biochem. J. 269, 393-402.

18.

19.

20.

70

Blumenkrantz, N. and Asboe-Hansen, G. (1973) Anal. Biochem. 54, 484-489.
Kindel, P. K. and Watson, R. R. (1973) Biochem. J. 133, 227-241.
Kindel, P. K., Cheng, L. and Ade, E. R. (1995) Phytochemistry, Vol. 41, No. 3, 719-

723.

CHAPTER IV

A MICRO-SCALE METHOD FOR DEPOLYMERIZATION

OF PECTIC POLYSACCHARIDES

71

72

ABSTRACT

A micro-scale method utilizing methanolysis, methylester formation, reduction and
hydrolysis was developed for depolymerizing pectic polysaccharides. The neutral
monosaccharides that were formed were converted to alditol acetates for analysis by gas
chromatography and mass spectrometry. For routine analysis, samples of 0.11-0.14 mg
were used and all reactions were performed in a single vial. The depolymerization of the
polysaccharides and the reduction of the carboxyl groups of the galacturonosyl residues

were virtually complete and sample recovery was good.

73

INTRODUCTION

When chromatographic methods such as GC and HPLC are used to determine the
sugar composition of a pectic polysaccharide the sample must first be depolymerized.
Typically this is done by acid hydrolysis or methanolysis. Problems with acid hydrolysis
are that the glycosidic linkage of uronic acid residues is quite resistant to acid hydrolysis
(1), and the released sugar acids decompose at a faster rate than the corresponding neutral
sugars (2). One way investigators have attempted to solve these problems is by reducing
the uronic acid residues to neutral sugar residues before hydrolysis. The method of Taylor
and Conrad (3), which involves the use of a water-soluble carbodiimide and the reduction
of a putative intramolecular ester by NaBH4, is widely used but has some disadvantages.
The method requires a relatively large amount of sample (0.1 mole of uronosyl units),
simultaneous treatment of multiple samples is difficult, sample loss occurs during the
desalting step and complete reduction is not obtained with a single treatment and
consequently the process must be repeated one or more times (3 -7). Anderson and Stone
(8) modified the method for use with smaller samples by using buffer to control the pH,
but our results with several pectic polysaccharides showed the buffer failed to maintain the
proper pH and complete reduction of carboxyl groups was not achieved. A method that
involved methyl esterification followed by reduction with LiAlH4 (9) or NaBH4 (10) was
used successfully to reduce the carboxyl groups of uronic acid residues of
oligosaccharides. However, it was not capable of completely reducing the carboxyl groups

of three pectic polysaccharides tested (this paper). De Ruiter et al. (1 1) described a

74

method involving methanolysis, TFA hydrolysis and the use of HPAEC-PAD to separate
and measure the amount of mono- and oligosaccharides produced from the
polysaccharides. The separation of mono- and oligosaccharides of galacturonic acid was
good with this method, but the separation of neutral monosaccharides was not. Since the
methylester of uronic acid residues that are formed during methanolysis should be quickly
hydrolyzed in the subsequent TFA hydrolysis step and released as free uronic acids, the
method could still result in almost as great a loss of uronic acid by decomposition as direct
acid hydrolysis of pectic polysaccharides. Consequently, it was not surprising to find that
sample recovery was low with the method (this paper). In this paper, we report a micro-
scale method that effectively depolymerized three pectic polysaccharides tested. The
method involves methanolysis/methyl esterification, reduction with NaBH4 (or NaBD4),
a repeat of these two steps, acid hydrolysis and preparation of the alditol acetates. The
entire procedure, including preparation of alditol acetates, was performed in a single

vessel.

MATERIALS AND METHODS

Materials. Apple pectic acid were obtained from Aldrich and PGUA was obtained
from Sigma. A cell wall pectic polysaccharide was isolated from Lemna minor; it had a
M., of 66,400, consisted of 97 mole% galacturonic acid and small amounts of apiose and
xylose, and had a degree of methyl esterification of less than 1.0%. A stock solution of

methanolic HCl (3.8 M) was prepared by dissolving the HCl gas generated from the

75

reaction of H2804 with NH4C1 in methanol. Immediately before diluting, the concentration
of HCl in the stock solution was redetermined by titration.

Depolymerization of pectic polysaccharides. Cell wall pectic polysaccharide from
L. minor, 0.4 mg, and polygalacturonic acid and apple pectic acid, each 0.5 mg (samples
of 0.11-0.14 mg were used in the routine analysis where only GC-MS analysis was
performed), were subjected to the following three depolymerization methods. Method 1:
The methanolysis and acid hydrolysis procedure of De Ruiter et al.(1l). Method 11: The
samples were methylesterified, reduced and acid hydrolyzed as described by Hollingsworth
et al.(10). Method 111 (proposed method): Dry samples were treated with 0.5 mL of 2 M
HCl in dry methanol and 0.05 mL of trimethylorthoformate at 80°C for 8 h with sonication
at 80°C for 5 min after 1 h. t-Butanol was added and the samples were dried in ambient
air with N2. The samples were reduced with 0.8 mL of NaBD4 in aq. 75 % (v/v) ethanol
(2 mg/mL) at 22°C for 10 h. The samples were acidified with glacial acetic acid (usually
two drops), mixed and dried with N2 at 30°C or less. Methanol (1 mL) was added and the
samples were dried at 30°C or less. Addition of methanol and drying was repeated four
times and the samples were desiccated in vacuo. The above procedure of depolymerization
and reduction was repeated once and the samples were hydrolyzed with 0.5 mL of 2 M
TFA at 110°C for 0.5 h. The hydrolyzed samples were analyzed by GC-MS (Appendix
B) after conversion of sugars to alditol acetates (12,13) and by HPAEC-PAD. The total
carbohydrate and uronic acid of the samples were measured before and after
depolymerization by the method of Dubois et al. (14) and Blumenkrantz and Asboe-Hansen

(15), respectively, and the results were used to calculate percent recovery of sample and

76

percent uronic acid remaining in the sample (16, Appendices C and D). Sample recover
is the sum of the mole of Api, Ga], and AUA after depolymerization divided by the sum
of the mole of Api, Ga], and AUA before depolymerization and times 100 (Appendix D).

Establishing conditions for Method 111. For the first methanolysis/methyl
esterification, 0.5 mL of 2 M HCl in methanol was added to vials containing 0.11-0.14
mg of cell wall pectic polysaccharide from L. minor. The samples were heated at 80°C
for the time periods shown in Table 4.2 and were dried-with N2 and reduced with NaBH4
as described above in Method 111. After reduction, the samples were hydrolyzed with 2
1M TFA for the time periods shown in Table 4.2 and dried with N2 at 30°C or less. The
second methanolysis/methyl esterification was with either 2 M or 0.18 M HCl/methanol
and time and temperature were varied as shown in Table 4.2. The samples then were
reduced with NaBH4 as in Method III. Additional information on optimum conditions was
obtained with apple pectic acid. Samples of 0.13 mg were treated as described in Method
111 except the time periods of both methanolysis/methyl esterification treatments were
varied as shown in Figure 4.1.

HPAEC—PAD. Depolymerized samples were dissolved in 1 mL of water and were
analyzed by HPAEC-PAD (Dionex Bio LC system, Dionex Corp). The HPAEC-PAD
system was equipped with a CarboPac PAl column (4 x 250 mm) and a CarboPac PAl
guard column and was operated at 22°C. The flow rate was 1 mL/min, samples of 20 aL
were injected, and the column was developed by using, in order, the following times and
eluents: 0-18 min, 20 mM NaOH; 18-45 min, a double gradient consisting of 20 to 100

mM NaOH and 0 to 1 M sodium acetate, both increased linearly; 45-55 min, the

77

concentration of NaOH and sodium acetate was kept unchanged; 55—60 min, the
concentration of NaOH and sodium acetate was decreased linearly to 20 mM and 0 M,
respectively. Chromatograms were recorded and integrated with both a HP3390 integrator
and a computer. The latter was connected to the HPAEC-PAD system through a SRI
interface and operated with PEAK-II software (SR1 Instruments, Torrance, California).
Standard samples of galactose and galacturonic acid were used to calibrate the HPAEC-
PAD system and to quantify these two sugar in samples.

Standard sugar mixture treated by Method 111. Standard mixtures containing 60 to
80 ,ug each of rhamnose, fucose, arabinose, xylose, apiose, mannose, PGUA, and glucose
were treated as follows: (i) 60 ug of myo-inositol was added (as an internal standard) and
the sample was treated by Method III and (ii) the standard sample mixture was treated by
Method 111 and then 60 ug of myo-inositol was added. In the preparation of the
peracetates (12,13) the following changes were made: (i) the quantity of reagents used was
2/5 that used by Kindel and Cheng (12) and (ii) the dichloromethane solution containing
the alditol peracetates was 0.6 mL and it was vigorously back-extracted with 3 mL of water
four times. The products were analyzed by GC (13, Appendix B). A response factor (GC

peak area/mass of sugar) for each sugar was calculated.

RESULTS

Comparison of Methods I, II and III. Samples were incompletely depolymerized

when Methods I and II were used since significant amounts of incompletely hydrolyzed

78

known oligogalacturonic acids and unknown products were detected (Table 4.1). The
unknown products that eluted in oligogalacturonic acids region were considered to be
galacturonic acids-containig oligosaccharides different than the oligogalacturonic acid
standards. For Methods I and II, the unidentified charged products accounted for 6.0—15 %
and 7 . 1-9.4 % , respectively, of the total product peak area detected with the three
polysaccharides tested. With Method HI, depolymerization of the three pectic
polysaccharides was virtually complete since no oligogalacturonic acids were detected and
the unidentified peaks in oligogalacturonic acid region were negligible (< 0.18 of the total
peak area, Table 4.1). The recovery of the sample after being depolymerized by Method
I was 48-54% , while the recovery of samples in Methods II and III was 77-90% and 80-
85%, respectively (Table 4.1). Although Method H gave a high recovery of sample, the
conversion of sugar acid to neutral sugar was very incomplete, as shown by the Gall GalA
mole ratios of between 10/90 and 20/ 80 for the products (Table 4.1). In contrast, carboxyl
reduction of the three pectic polysaccharides analyzed by Method III is virtually complete,
since the mole ratio, Gal/GalA, in the depolymerized samples was 97-98/2.9-2.1 (Table
4.1). For all three methods and samples tested, peaks of unknown compounds were also
detected by HPAEC-PAD in the region where neutral sugars eluted. The peaks in neutral
sugar region were not identified but were also observed in the GalA control. These peaks
are not listed in Table 4.1.

Optimization of conditions for Method 111. When Method II] was used but the second
methanolysis was conducted with 0.18 M HCl in methanol at 45°C for 2 h and the samples

were hydrolyzed with 2 M TFA at 110°C for 0.5, 1.0 and. 1.5 h before second

79

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methanolysis, the uronic acid remaining in the sample after reduction was 21 % , 19% and
16% (Table 4.2). When the same conditions were used but the time of the second
methanolysis was increased to 14 h plus 10 h at 22°C, the uronic acid remaining only
decreased from 19% to 14% (l h TFA samples, Table 4.2). When the temperature of the
second methanolysis was increased from 45°C to 80°C and methanolysis times of 2 and
4 h were used, 16 and 14% ,respectively, of the uronic acid residues still remained in the
samples (Table 4.2). However, when the concentration of HCl/methanol in the second
methanolysis was 2 M and each methanolysis was for 1 h at 80°C, the uronic acid
remaining in the sample was 5.6 % (Table 4.2). Further reduction of the remaining uronic
acid residues was difficult. When the times of the first and second methanolysis were
increased to as much as 15 and 8 h, respectively, the uronic acid residues remaining in the
sample still did not decrease below 3.6% (Table 4.2). A time course experiment
examining the depolymerization of apple pectic acid showed that there was only a slight
decrease in the uronic acid content of the depolymerized samples when the two
methanolysis times were increased from 1.5 h : 1.5 h to 10 h : 10 h (Figure 4.1). At the
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methanol showed this hydrolysis was not necessary (Table 4.2) and it was eliminated from
Method 111.

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82

Figure 4.1 . Galacturonic acid remaining after depolymerization of apple pectic
acid by Method III used with varying methanolysis times Times of the first
methanolysis are indicated in the figure. Times of the second methanolysis were: 0.25
h (—-D—), 0.75 h (---A---), 1.5 h (---o---), 3 h (---C---) and 10 h (--I|I--). GalA remaining

(%) after depolymerization was calculated as described in the Materials and Methods.

GalA remaining (%)

l2.5

83

 

 

 

 

 

 

u—d—

Time of first methanolysis (h)

10

ll

84

Figure 4.2. GC response factors of eight alditol peracetates and myo-inositol
peracetate (as internal standard) based on area/mg of sample. Response factors of
alditol acetates are relative to myo-inositol which was set to unity. Response factors of
alditol peracetates when myo-inositol was added after E3 and before the sugars were
treated by Method 111. Response factors of sugars not treated by Method 111 but

hydrolyzed with 2 M TFA at 120°C for 1 h and converted directly to alditol peracetates

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86

researchers to hydrolyze neutral polysaccharides. Addition of internal standard (myo-
inositol) before and after depolymerization by Method III did not have a significant effect

on the response factors.

DISCUSSION

In our proposed method the pectic polysaccharides were partially depolymerized and
methylesterified by the first methanolysis and then the esterified carboxyl groups were
reduced. The hydrolysis with TFA cleaved the remaining mainly reduced oligosaccharides
as well as the methyl glycosides. Although the total time of methanolysis (16 h) in our
proposed procedure is the same as in Method 1, the second methanolysis was conducted
after most of the uronic acid residues were reduced to neutral sugar residues. Since the '
glycosidic linkages of neutral sugars are much more susceptible to acid hydrolysis than
those of uronic acids (1) and since the released neutral sugars are less susceptible to acid
degradation than are uronic acids (2), the conversion of uronic acid to neutral sugar in our
procedure both enhanced the depolymerization of the pectic polysaccharides as well as
reduced the loss of released sugar during both the methanolyses and the TFA hydrolysis.
The procedure resulted in complete depolymerization of polysaccharides (no
oligosaccharides were observed) and a much higher sample recovery (80-85%) than the
procedure of De Ruiter et al. (11) (48-54%), at least for the three pectic polysaccharides
tested (Table 4.1).

Methyl esterification with 0.18 M HC] in dry methanol at 45°C is rigorous enough to

87

make the methylester of small oligosaccharides that contain some uronic acid residues (10)
but is not adequate for methylesterifying the pectic polysaccharides and partially
depolymerized pectic polysaccharides tested since the mole ratio, Gal/GalA, in the final
product in Method 11 ranged from 20/ 80 to 10/90 (Table 4.1). After a first methanolysis
with 2 M HC] in methanol, increasing the temperature of methyl esterification in the
second methanolysis from 45 °C to 80°C or extending the time of the treatment to 14 h plus
10 h at 22°C did not substantially improve methyl esterification since the GalA remaining
in samples after depolymerization decreased only from 19% to 16% and 14% (Table 4.2).
Strengthening the methanolysis/methyl esterification conditions from 0.18 M to 2 M HC]
in dry methanol and 80°C resulted in more complete methyl esterification since the ratio
of Gal/GalA now ranged from 98/2 (Table 4.1) to 96/4 (Table 4.2) even though the time
of both the first and second methanolysis/methyl esterification was as short as 1 h each.
This indicated the concentration of H Cl/methanol was more important than temperature
or time of methanolysis in the conversion of GalA units to Gal. Since the glycosidic
linkage to neutral sugars is more susceptible to methanolysis and acid hydrolysis than is
the same linkage to uronic acids, it is not surprising that virtually complete reduction of
the galacturonic acid residues in Method III resulted in almost complete depolymerization
(Table 4.1).

Acetylation of eight monosaccharides with or without first treating the sample by
Method III did not result in significant change for the GC response factors for alditol
acetates. These results indicated Method 111 (i) did not interfere with the followed

. acetylation process and (ii) did not cause significant sample loss of the tested sugars.

88

Addition of myo-inosito] before or after Method III did not resulted in significant change
the GC responce factor of myo-inositol compared to those of sugar alditol acetates; this
showed Method 11] did not cause significant loss of myo-inositol.

Although the separation of oligogalacturonic acids with a degree of polymerization up
to 15 with the HPAEC-PAD system is fast and complete (data not presented), the
separation of the eight neutral monosaccharides examined by this system is incomplete.
Complete separation of the common alditol acetates and the ease of interfacing a gas
chromatograph to a mass spectrometer still make GC-MS a more accurate system than
HPLC-MS for sugar composition analysis and identification in most cases. The
advantages of the proposed depolymerization method over others in use (3,11) are: (i)
more complete depolymerization of pectic polysaccharides, (ii) greater conversion of
uronic acid residues to neutral sugars, (iii) reactions are performed in a single vial and
dialysis is not necessary, (iv) 0.11-0.14 mg or less of sample is needed, (v) multiple

samples can be treated simultaneously and (vi) recovery of sample is higher.

10.

11.

12.

13.

14.

89

REFERENCES

BeMiller, J. N. (1967) Adv. Carbohydr. Chem, 22, 25-108.

Biermann, C. J. (1988) Adv. Carbohydr. Chem. Biochem., 46, 251-271.

Taylor, R. L., and Conrad, H. E. (1972) Biochemistry, 11,1383-1388.

Schols, H. A., Posthumus, M. A. and Voragen. A. G. (1990) Carbohydr. Res.
206,117-129

Kiyohara, H., Cyong J. C. and Yamada, H. (1988) Carbohydr. Res., 182, 259-
275.

Saulnier, L., Brillouet, J. M. and Joseleau, J. P.(1988) Carbohydr. Res.182, 63-78
Lan, J. M., Michael, M., Darvill, A. G. and Albersheim, P. (1985) Carbohydr.
Res., 137, 111-125.

Anderson, M. A. and Stone, B. A. (1985) Carbohydr. Polymers, 5,115-129.
Adams, G. A. and Bishop, C. T. (1956) J. Am. Chem. Soc., 78, 2842-2844.
Hollingsworth, R. 1., Abe, M., Sherwood, J. E. and Dazzo, F. B. (1984) J.
Bacteriol., 160, No. 2, 510-516.

De Ruiter, G. A., Schols, H. A., Voragen, A. G. J. and Rombouts, F. M. (1992)
Anal. Biochem. 207, 176-185.

Kindel, P. K. and Cheng, L. (1990) Carbohydr. Res., 199, 55-65.

Cheng, L. and Kindel, P. K. (1996) Carbohydr. Res., 290, 67-70.

Dubois, M. G., Gilles, K. A., Hamilton, J. K., Rebers, P. A. and Smith, F. (1956)

Anal. Chem, 28, 350-356.

15.

16.

17.

9O
Blumenkrantz, N. and Asboe-Hansen, G. (1973) Anal. Biochem., 54, 484-489.
Kindel, P. K., Cheng, L. and Ade, B. R. (1996) Phytochemistry, Vol. 41, No. 3, 719-
723.
Albersheim, P., Nevins, D. J ., English, P. D. and Karr, A. (1967) Carbohydr. Res.,

5, 340-345.

CHAPTER V

HOMOGENEITY AND STRUCTURE OF THE 22°C AMMONIUM OXALATE-

SOLUBLE PECTIC POLYSACCHARIDES OF Lemna minor

[Adapted from Cheng, L. and Kindel, P. K., Carbohydr. Res., 1996, submitted]

91

92
ABSTRACT

The homogeneity of pectic polysaccharides from Lemna minor purified through DEAE-
Trisacryl Plus M column chromatography was determined by analyzing the polysaccharide
material in individual column fractions. Determining the MW of such polysaccharide material
by using multi-angle laser light scattering detector in conjunction with high performance
size exclusion chromatography showed that four pectic polysaccharides were present in the
22°C ammonium oxalate-soluble fraction prepared from purified cell walls of L minor. The
four pectic polysaccharides were designated PS-I, -II, -III and -IV and the peak M“, ranges
of the purified polysaccharides were: PS-I, 50,200 — 75,400; PS-II, 18,800 - 99,700; PS-III,
24,200 - 150,000; and PS-IV, 6,170 to at least 163,000. PS-IIb and PS-IVb, major
components of PS-H and PS-IV, respectively, were basically homogeneous with respect to
sugar composition and degree of methyl esterification, each however was different
chemically. The average sugar composition of the polysaccharide material analyzed that
constituted PS-IIb was: GalA (54.5:3.2%) (std. dev.), Api (39.3 :I: 1.4%), Xyl (2.5% 1:] .4%),
Ara (1.4% :t 0.8%), and Rha (2.3 t 0.3%) while that of PS-IVb was: GalA (96.3% :t 0.8%),
Api (2.1 :t 0.7%), and Xyl (1.6 i 0.3%). Small amounts (<0.5%) of other common sugars
were detected in the two polysaccharides. The degree of methyl esterification of both PS-IIb
and PS-IVb was basically zero. The isolation of pectic polysaccharide of high galacturonic
acid content (96%) and low degree of methyl esterification (~0%) while using mild
conditions is new. PS-IIb and PS-IVb were heterogeneous with respect to size of molecules.
The peak MW range of PS-IIb was 28,100 - 99,700 while the range for PS-IVb was 53,300

g to at least 163,000. A simple, mild procedure for quantitatively isolating and purifying pectic

93

polysaccharides from the 22°C ammonium oxalate-soluble fraction was deve10ped.

INTRODUCTION

Pectic polysaccharides constitute one of the three major polysaccharide components of
primary cell walls of plants (1). It is well known that a portion of the cell wall pectic
polysaccharides is solubilized by chelating agents at room temperature. Recently, the
kinetics of the solubilization process were reported (2) (Appendix A). Kinetic experiments
with L. minor (duckweed) and Apium graveolens (celery) showed that solubilization of the
22°C ammonium oxalate-soluble fraction occurs rapidly and is almost complete in 15 min
and that further solubilization virtually ceases after 30 min (Appendix A). With purified cell
walls of fresh L minor and store-purchased A. graveolens, 32% and 22%, respectively, of
the total anhydrouronic acid of the cell wall is solubilized in 30 min. These results show that
on the basis of solubility, the pectic polysaccharides of the 22°C chelator-soluble fraction are
different from the pectic polysaccharides remaining in the cell wall, at least in these two
plants. Whether they also differ structurally is not yet known.

Purification and characterization experiments indicate that the 22°C chelator-soluble
fraction consists of more than one component (3-11); however, the results do not rule out the
possibility that there is a continuum in the structure of the pectic polysaccharide material in
the 22°C chelator soluble fraction and discrete polysaccharides are not present. Typically
the last step in the purification of pectic polysaccharides from this fraction is DEAE column

, chromatography. The homogeneity of the pectic polysaccharides eluted from the columns

94

has not been established. The goal of this study was to determine the degree of homogeneity
of the isolated pectic polysaccharides and then determine their structure. Determining the
degree of homogeneity first will simplify the interpretation of structural results. Homogeneity
was established by determining different pr0perties of the pectic polysaccharide material in
the individual DEAE column fractions and then comparing each property across column
fractions. If a property of the pectic polysaccharide material in a contiguous set of fractions
was the same, the pectic polysaccharide material in these fractions would be homogeneous
with respect to that property. The M.,, sugar composition, and degree of methyl esterification
were the properties determined in this study. Appropriate fractions were also combined as
individual polysaccharides for further analysis. The pectic polysaccharides were isolated
from the 22°C ammonium oxalate-soluble cell wall fraction of L minor. Since the recovery
of pectic polysaccharide material from the DEAE column was quantitative, all the pectic
polysaccharides in the fraction were examined. A second goal of the work was to simplify
the purification procedure of pectic polysaccharides from cell walls while still maintaining

non-degradative conditions.

MATERIALS AND METHODS

General method. L minor was grown as described elsewhere ( 12). Total carbohydrate
and uronic acid were determined by the methods of Dubois et al. (13) and Blumenkratz and
Asboe-Hansen (14), respectively. DEAE-Trisacryl Plus M, polygalacturonic acid and citrus

. pectin were purchased from Sigma Chemical Co. Apple pectic acid and D-galacturonic

95

acid-H20 were purchased from Aldrich Chemical Co. and Pfanstieh] Laboratories, Inc.,
respectively. Information on the above three polysaccharides has been given previously (15).

Isolation of pectic polysaccharides. Cell walls were isolated from fresh L minor (597
g) as described by Kindel et al. (Appendix A and Ref. 2). The cell walls were extracted
immediately with 1070 mL of 0.05 M ammonium oxalate (pH 5.5) at 22°C for 30 min, and
the suspension was filtered with 15 um Nylon mesh (3-15/6, Tetko, Inc.). The cell wall
residue was resuspended in 850 mL of 0.05 M ammonium oxalate (pH 5.5), swirled several
minutes, and the suspension was filtered. The combined filtrates were filtered successively
with 5 and 1.2 pm membrane filters (MP-Millipore, Millipore Corp.). A small portion of the
final filtrate was removed for determination of uronic acid. The remainder was applied
directly to a DEAE-Trisacryl Plus M column, 2.9 cm (i.d.) x 24 cm (height), and the column
was washed with 200 mL of 0.05 M ammonium oxalate (pH 5.5) (column buffer) and then
developed with 4800 mL of column buffer containing a linear gradient of 0 to 0.5 M NILC].
Fractions of 20 mL were collected at 1.06 mIJmin and analyzed for uronic acid. The MW,
sugar composition, and degree of methyl esterification of the polysaccharide material in
selected column fractions were determined.

Rechromatography of pectic polysaccharides with DEAE-Trisacryl Plus M. Portions
(0.5 mL) of DEAE-Trisacryl column fractions 195-224 and 287-320 (fraction numbers refer
to Figure 5.3 in Results) were combined to give two polysaccharide samples called PS-IIb
and PS-IVb, respectively. PS-IIb and PS-IVb were dialyzed in water, ammonium oxalate
was added to each to a final concentration of 0.05 M and the pH of the solutions was

. adjusted to 5.5 with oxalic acid. Each sample was applied to a separate column of DEAE-

96

Trisacryl Plus M [0.7 cm (i.d.) x 8 cm (height)]. Each column was washed with 0.05 M
ammonium oxalate (pH 5.5) (column buffer), and developed with 100 mL of column buffer
containing a linear gradient of 0 to 0.5 M NH4C1. Fractions of 1 mL were collected at 3.6
mIJh and were tested for uronic acid.

Refractive index increment (dn/dc) of polysaccharide samples. The differential
refractometer (Knauer, km from a light-emitting diode = 950:1:30 nm) was calibrated with
NaCl. The Cauchy dispersion formula (terms of higher order than B/lt2 were not used) was
used to extrapolate the refractive index data of Kruis (16) at 9 concentrations of NaCl
(0.09412 to 6.9092 g/ 100 g H20) to 950 nm. A plot of (n-no)”Onm (from extrapolation) versus
concentration of NaCl was prepared, and the following polynomial in concentration was
obtained:

(n—no)950 m x 10° = 0.65345C3 - 21.987C2 + 1680.6C + 2.6472
where (n-n°)950 “m is the difference in refractive index between solution (n) and solvent (no)
at 950 nm and C is the concentration of NaCl in g/ 100 g H20.

The instrument relative refractive index (RRI) reading of 5 concentrations of NaCl
(0.0056, 0.0190, 0.0571, 0.1942 and 0.5009 g/ 100 g H20) that were passed through the
differential refractometer at 2 mI/min and the (n-no)950 “m of these 5 concentrations calculated
from the above polynomial were plotted separately against the concentration of N aCl (Figure
5.1). Because the change in slope of the line was so small over the concentration range of
NaCl used, linear regression analysis of the data was performed. The slope of the former
plot, (dRRI/dc)NaCl = 2026.8 RRI units (g NaCl/100 g H20)“, and the slope of the latter,

.(dn/dc)N,C, = 1669.9 x 10*5 (g NaCl/100 g H20)", were used in the equation below to

97

Figure 5.1. Relationship between RRI reading ( A ) or calculated refractive index at
950 nm (n—no),,o m ( O ) and concentration of NaCl. The calculation of (n—no)950mm and

the linear regressions are described in the Materials and Methods.

98

 

1250 1250

1000_ _1000
y = 2026.8x + 2.4 ( A )

750- - 750

RR] reading

y =1669.9x + 3.0 (o)
250- - 250

 

 

 

NaCl (g/ 100 g water)

106 X (n ’ no)950 nm

99

determine the refractive index increment of pectic polysaccharide samples.

A portion (2 mL, 0.53 - 0.83 mg of pectic polysaccharide) of each of fractions 222, 223,
284 and 290 from the DEAE column chromatography (fraction numbers refer to Figure 5.3
in Results and samples were called polysaccharides F222, F223, F284 and F290,
respectively; polysaccharide material in other column fractions were named similarly) was
dialyzed against 0.35 M NH4C] (column buffer) for 28 h with dialysis tubing having a
molecular weight cut-off of 6000-8000. The dialyzed samples were passed through the
differential refractometer at a rate of 2 mI/min. The relative refractive index readings were
obtained with 0.35 M NH4Cl as the reference and the sample dry weights were calculated
from total carbohydrate and uronic acid determinations. The (1de values of the
polysaccharides were calculated with the equation:

(dRRI/dC)NaCl/(dn/dC)NaC] = (dRRI/dCholysaccmdc/(dn/ddpo.mama:

where (dRRI/dc)polysaccharide is the slope from linear regression analysis of the data in the plot
of relative refractometer reading versus concentration of the pectic polysaccharide tested, and
(dn/dc)polysaccharide is the refractive index increment of the polysaccharide. The (1de values of
the four pectic polysaccharides and polygalacturonic acid are given in Table 5.1. Since the
(1de of the polysaccharide material in every column fraction examined by MALLS (multi-
angle laser light scattering) was not determined, appropriate (1de values of Table 5.1 were
averaged and average values were used to calculate the M.,, of polysaccharides.

Determination of A7,, and HPSEC. The M , M and polydispersity of the

polysaccharide(s) in DEAE column fractions 189, 207, 214, 217, 220, 223, 227, 270, 278,

282, 284, 286, 288, 290, 294, 296 and 298 of the DEAE column chromatography (fraction

100

Table 5.1. Refractive index increment of pectic polysaccharides

 

 

Sample (1de
polygalacturonic acid 0.1757
F222“ 0. 1072

F2238 0. 1088

F284a 0. 1526

F290“ 0. 1784

 

 

 

 

aPolysaccharide material in correspbnding DEAE column fractions of Figure 5.3.

101

numbers were referred to Figure 5.3 in Results) were determined with a miniDawn MALLS
detector (Wyatt Technology Corp.) used in conjunction with an in-line differential
refractometer and an in-line HPSEC column [G4000PWXL, 7.8 mm (i.d.) x 30 cm (height),
particle size: 10 um] (TosoHaas). Centrifuged samples of 100 pL from the above DEAE
column fractions were chromatographed on the G4000PWXL column shortly after being
collected and before being frozen. The column was developed with 0.35 M NH4C1 at a rate
of 0.6 mUmin. HPSEC solvents were filtered through a 0.22 pm filter (Durapore-
hydrophilic, Millipore Corp.). The light scattering and refractometry signals for
polysaccharide material eluting from the column were collected by a computer (IBM
compatible PC). The refractometry signal was also recorded with an integrator (Model
3390A, Hewlett-Packer Co.). The computer data were processed with ASTRA 4.0 and EASI
software (Wyatt Technology Corp.) and MW, Min, and polydispersity and peak MW and peak
M, were calculated. Peak M“, is defined as the MW, determined either directly from light
scattering data or indirectly from the two equations of linear regression described below, of
a narrow band of polysaccharide material at the top of the polysaccharide peak (band) eluted
from the HPSEC column, with polysaccharide being detected by refractometry (see Figure
5.4 of Results). Peak IV]w values were calculated because retention times from HPSEC were
used to calculate 1V1.w values of samples not analyzed by MALLS.

Two different linear regression equations of K“ [Kw = (Vc - Vo)/(V. - V.,): where V,,Vo
and Vt were elution volumes of pectic polysaccharide, Dextran T-2000, and D-xylose,
respectively, determined by HPSEC] versus peak lVIw (from MALLS) were established and

used to calculate the peak M., of column fraction samples (Figure 5.2). Equation A (Figure

102

Figure 5.2. Relationship between Kav and Log IVIw for those pectic polysaccharide
fractions analyzed by HPSEC and MALLS. The polysaccharide fractions used to obtain
linear regression equations A and B are given in the Materials and Methods. The calculation

of MW is described in the Materials and Methods.

Kav

0.70

103

 

0.60 -

    

0.50 a

0.40 -

0.30-

0.20-

 

0.10

A: y : -0.527x + 2.920 ( E] )

 

 

4.25 4.50

4.75 5.00 5.25

Log Peak MW

104

5.2) was derived from the results of HPSEC and MALLS for F189, F207, F214, F217, F220,
F223, F227, and F270 and was used to determine the peak MW of the polysaccharide
materials in the indicated column fractions of Figure 5.3 that were part of PS-I, PS-II, and
PS-III. Equation B (Figure 5.2) was derived from the results of HPSEC and MALLS for
F278 (band 2), F282 (band 2), F-284 (band 2), F286, F288, F290, F294, F296, and F298 and
was used to determine the peak MW of the polysaccharide material in the indicated column
fractions of Figure 5.3 that was part of PS-IV. The selection of data used to establish linear
equations A or B in Figure 5.2 was based on that the plotted values in the figure and were
most appropriately represented by the two linear equations. PS-I through PS-IV and bands
1 and 2 are defined in the Results.

Depolymerization of pectic polysaccharides and preparation of alditol acetates. Pectic
polysaccharide samples (approximately 0.14 mg dry weight) were dialyzed against water,
freeze-dried, depolymerized and converted into adito] acetates as described in Chapter IV of
this thesis.

Gas chromatography and mass spectrometry. Alditol acetates were separated
isotherrnally at 230°C on a DB-225 fused silica capillary column (30 m x 0.25 mm, i.d.; film
thickness, 0.15 pm) (J &W Scientific) attached to a gas chromatograph, Model 5840A
(Hewlett-Packard Co.), equipped with a splitter, a flame-ionization detector and a 5840A
data terminal. Helium was the carrier gas and the flow rate was 1.45 mI/min. Samples
injected into the gas chromatograph ranged from 0.5 to 2.5 uL and the split ratio ranged from
1:10 to 1:20. The FID signal was recorded with a computer (IBM compatible PC) through

- a interface A/D board (Model 110 BUS, SRI Instrument) and processed by PeakSimple II

105

software (SRI Instrument).

Electron impact mass spectrometry of alditol acetates was performed with a JEOL J MS-
HXl 10, double-focusing mass spectrometer (JEOL USA) interfaced to a gas chromatograph,
Model 5890A (Hewlett-Packard Co.), which was equipped with a splitless injector and a DB-
225 fused silica capillary column (30 m x 0.32 mm, i.d.; film thickness, 0.25 pm). Helium
was the carrier gas and the flow rate was 4 mI/min. The proportion of galactose units to
galacturonic acid units in samples was determined by selective ion monitoring. The ratio,
6,6-dideuteriogalactitol acetate/6,6-dideuteriogalactitol acetate + galactitol acetate, was the
mean of the ratios calculated from two ion clusters: (i) ions m/z 217, 218 and 219 and (ii)
ions m/z 289, 290 and 291 (Appendix E). Standard galactitol acetate was prepared from D-
galacturonic acid-H20 and NaBD4 at the same time samples were prepared. The value of the
ratio for the standard was set to 100% and the samples were adjusted accordingly.

Determination of degree of methyl esterification. The procedure of Wood and
Siddiqui (17) was used to determine the methyl ester content of polysaccharide samples. The
degree of methyl esterification is the molar ratio of methyl ester/uronosyl unit for each
sample, expressed as a percent.

1H NMR spectroscopy. Pectic polysaccharide material from column fractions 289, 290
and 292 of Figure 5.3 was dialyzed against water and adjusted to pH 6.0. For F289 and F292
the pH adjustment was by evaporation and addition of D20 4 times and then addition of
NaOD in D20. F290 was adjusted with NaOH before evaporation and then subjected to the
same evaporation and D20 addition as F289 and F292. The pH of F290 remained at 6.0.

. Polygalacturonic acid and apple pectic acid in water were adjusted to pH 5.0 with NaOH,

106

lyophilized and approximately 5 mg of each was dissolved in 0.8 - 1.0 mL of D20. F223 was
treated the same except no pH adjustment was made. The samples were analyzed with a
VXR-SOO NMR spectrometer (Varian Instruments) at 90°C. The reference frequency was
the HBO (in D20) peak at 3.937 ppm. In a separate analysis, the HDO reference frequency
(3.937 ppm) was obtained by reference to a p-dioxane peak which was locked at 3.530 ppm
at 90°C.

RESULTS

Isolation of pectic polysaccharides. The pectic polysaccharides solubilized from
purified cell walls by 0.05 M ammonium oxalate at 22°C were chromatographed on a
DEAE-Trisacryl Plus M column. The column chromatogram, based on determination of
uronic acid, is shown in Figure 5.3. Recovery of uronic acid from the column was 99.4%.
Although not determined in this experiment, recovery of total carbohydrate in analogous
experiments was also quantitative and the total carbohydrate elution profile had the same
general shape as the uronic acid profile. Uronic acid and total carbohydrate (in previous
experiments) were not detected in column fractions 1 through 110. Two large peaks, a small
peak and a small shoulder on the rising side of the second large peak can be seen in the
uronic acid profile (Figure 5.3).

M}, 117,, and Polydispersity. The M.,, Mn, and polydispersity of the polysaccharide
samples that were analyzed directly by MALLS are given in Table 5.2. The M.,, M, and
polydispersity for the peak region of these samples are also given in Table 5.2. These peak

- lVlw values are plotted in Figure 5.3 as well so they can be compared to the peak IVIw values

107

Figure 5.3. Column chromatogram of the 22°C ammonium oxalate-soluble fraction
from purified cell walls of L minor. The column was DEAE-Trisacryl Plus M. Column
operation and determination of uronic acid ( O ) were as described in the Materials and
Methods. Salt concentration ( ----- -) was measured with an in-line conductivity meter. Peak
MW values for the four pectic polysaccharides calculated from the plots, Km, versus peak MW,
are designated as follows: PS-I ( O ), PS-II ( A ), PS-III ( I ), and PS-IV ( O ) with peak MW
values for the same polysaccharide connected by a dotted line ( ------------- ). Peak MW values
obtained directly from MALLS are designated by ( i- ). The vertical dotted lines and double-

headed arrows ( H ) designate the column fractions constituting PS-IIb and PS-Nb.

 

 

 

 

 

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240 260 280 300 320 340

Fraction number (20 mL/fraction)

109

fin 035. mo cam"— cca van— no 02.6 omfio><u

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038:3“ 82» fin 03E. mo gum can .38 .38 mo 035 omfio><o 82:05 a 33 _ Ban moi—5m 80:. E.
w _ 6 a5.— ooNE _ 089 _ 934 SEN— cooomm wt 6 woun—
mo; o8.— oonA ~ coug— wgg 83m: 88mm «2 .o can
3d 304 oomoa emwna mg; 02.8— 822 3:6 38
end moo; 82h cmchm ~ _ _.m 2 05 8.83 wt .o cog
mad 5o; 83% 2mg 3 mg ova: omwmh new _ .o wwfim
vmd moo; emmhm 88m awn; com: 235 $26 exam
and moo." 85mm 2.5mm So.— oowmm omv; mfld .N 9.3 vwam
mm; N8." o3 Hm omfim moo; 30mm 85mm mm _ .o .N 9.3 SE
:6 moo; Ago—Nu 2 «mm >8.— 3 _ mm Swen mm _ .c .N 2:3 mam
odd 03.— ovmcm omfim no: 958 on—R mad N 933 v53
med So.— oovufi cova mew; canoe 8~N2 cam—d _ 938 2:5 org
de SA: oomhm Sham one; 2.9m omocn pwomd Ram
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aod So; 0mm: 083. 2:.— ovaan 28w nus—d Guam
bad So; Some cacao :: ._ 2.08 82:. awn: .o S um
um; 25.— Omvwv oawwv mm ~ ._ cacow carom awe .o 3 mm
m _ .o as; Oman ovam van; Ono; one; awed 88
one «we; 8me 03mm mac; 8ch cvmmv awofio on E

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5:38 mam—Q 2: 89¢ 8383 «65383.23 95qu vogue—um .3 Aug fifiuonmmebcn ES :5 .35 2:. .N.m «Bah.

 

110

for these samples that were calculated from equations A and B (Figure 5 .2). These latter
peak l\7lw values are also plotted in Figure 5 .3. The peak MW values for all other samples are
also reported in Figure 5.3. These, of course, were calculated by using equations A and B
(Figure 5.2).

In all of the samples examined by MALLS some aggregated pectic polysaccharide
material was observed (Figure 5.4a). The aggregated material was readily detected by a light
scattering but the mass was too small to be detected by refractometry. The material present
in the aggregated form ranged from 0.18% to 1.9% of the total mass in the sample for all
samples except polysaccharide F189, which was 4.50% due to low sample concentration and
a high ratio of noise/signal (Table 5.2). Excluding the aggregated material, the elution
profiles from light scattering and refractometry of all the samples shown in Table 5.2, except
F227, F270, F278, F282, and F284, each had a single peak, indicating the samples were
homogeneous (Figure 5.4a). The light scattering and refractometry profiles showed that
samples F270 (Figure 5.4b), F278, F282 and F284 consisted of at least two polysaccharides
that were not completely separated. F227 migrated as a single band on HPSEC but the
increase in broadness of the band compared to F223 and the large change in IVIw between
F227 and F225 — which resulted in F227 being part of PS-III and F225 being part of PS-II
(see next section) — indicated that F227 consisted of more than one polysaccharide.

The profile areas used to calculate the IVIW and MD of the samples in Table 5.2 were based
on the refractometry profiles. For a majority of the samples this excluded the aggregated
material; however, for samples F288-F298 there was some overlap of aggregated material

with material detected by refractometry. Although the overlap increased the MW values of

1]]

Figure 5.4. HPSEC column chromatograms of L. minor pectic polysaccharides F217
(a) and F270 (b). Column operation is described in the Materials and Methods
Polysaccharide was detected by MALLS (I I I I) and refractometry (+ + + +). For F217,
bands between arrows are: (i), the entire sample including aggragation used to calculate MW;
(ii), region used to calculate the aggragate material in the sample; (iii), region used to
calculate peak M of the sample. For F270 (i) and (ii) are as for F217; (iii) and (iv) are the
regions used to calculate peak B71w of the two polysaccharides, PS-l]1 and PS-IV, respectively,

in the sample.

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113

these samples, the basic trend of increasing MW values across these samples was not altered.
For samples F270, F278, F282 and F284 the refractometry areas used for calculating 1171w
were selected either on the basis of the point minimum refractometry response between peaks
(F270) or where the refractometry response changed sharply but only gave a shoulder (F278,
F282, F284).

HPSEC. The polysaccharide material in each of column fractions 154 - 164 (Figure
5.3) migrated as a single band on HPSEC indicating a single pectic polysaccharide was
present. The peak M“, of the polysaccharide material in these fractions increased from
fraction 154 through 164. The polysaccharide material in each of column fractions 167-187
consisted of at least two pectic polysaccharides as indicated by the presence of two peaks on
the HPSEC column chromatograms (chromatograms not shown). These results indicated
two different pectic polysaccharides had eluted from the DEAE column. The first to elute
was called PS-I, the second, PS-II (Figure 5.3). In any DEAE column fraction containing
both, the VC of PS-I on HPSEC was always smaller than the Vc of PS-II. The Vc values of
both polysaccharides continually decreased from column fraction 167 through 187 and this
is reflected in their generally increasing peak 1171w values across these fractions (Figure 5 .3).
In fractions followed DEAE column fraction 187, PS-I was no longer detectable by HPSEC.
The polysaccharide material in column fractions 196-223 migrated as a single band on
HPSEC. The peak L71“, values of the polysaccharide material in these fractions linked
smoothly with the peak MW values for PS-II material in column fractions 167-189 therefore
the polysaccharide material in fractions 196-223 is considered to be part of PS-II (Figure

5.3). PS-IIb, described earlier, contained the majority of the mass of PS-II but did not

114

contain detectable amounts of PS-I.

The polysaccharide material in column fractions 225 and 227 was considered to be
different because of the substantial change in peak MW from 94,000 for the material in
column fraction 225 to 30,000 for the material in column fraction 227 and from the fact that
the peak MW of the polysaccharide material in the column fractions immediately preceding
225 was about 97,000 while the peak M., of the material in the column fractions immediately
following 227 was about 27,000. Two components may have been present in column
fractions 225 and 227 but a separation by HPSEC was not observed. For DEAE column
fractions 229 - 242, a single pectic-polysaccharide appeared to be present as indicated by the
single peak on the HPSEC column chromatogram; however, the peak was broad. The peak
1171,, values of the polysaccharide material in the fractions are given in Figure 5.3. As is seen
in Figure 5.3, the values linked together smoothly and progressively increased across the
fractions. Since the peak MW values of the pectic polysaccharide material in the initial
fractions of column fractions 229 - 242 was so much lower than the peak MW values of the
PS-II material in column fractions 222 and 224, the polysaccharide material in column
fractions 229 - 242 was considered to be part of a third pectic polysaccharide, called PS-III.

The polysaccharide material in each of column fractions 248-274 also consisted of two
pectic polysaccharides as indicated by the presence of two peaks on the HPSEC column
chromatograms. The situation was similar to what was found in fractions 167 - 187. The
polysaccharide material in column fractions 248 - 274 that eluted first from the HPSEC
column had peak MW values that linked smoothly with those of the PS-III material in column

, fractions 227 - 242 and therefore was considered to be the remainder of PS-III (Figure 5.3).

115

The polysaccharide material in these fractions that eluted second from the HPSEC column
was considered to be a fourth pectic polysaccharide, called PS-IV, and the peak L71“, values
of the PS-IV material in these fractions linked smoothly with the peak MW values for PS-IV
material in DEAE column fractions 276 - 305 (Figure 5.3). As stated earlier, aliquots of
column fractions 287-320 were combined to give PS-IVb, which did not contain detectable
amounts of PS-III. HPSEC chromatograms showed that PS-III material was visible as a
shoulder on the PS-IV peak until DEAE column fraction 284.

The results in Figure 5.3 show that four pectic polysaccharides were present in the 22°C
ammonium oxalate-soluble cell wall fraction of L. minor. Their peak 1171“, ranges were:
50,200-75,400; PS-II, 18,800 - 99,700; PS-III, 24,200 - 150,000; and PS-IV, 6,170 to at least
163,000. The results in Table 5.2 show that peak MW values are only slightly less than the
L7,, values for the entire sample for most of the samples constituting PS-II and PS-III. The
differences are greater for those samples that are part of PS-IV. Consequently the M., ranges
when the entire band of each sample is used will be somewhat higher than the peak 1V1,”
ranges stated above. IVIw values of samples constituting PS-I were not determined because
of insufficient quantity of sample for MALLS-refractometry analysis.

Sugar composition of polysaccharides. The sugar composition of the polysaccharide
material in selected column fractions of Figure 5.3 was determined and the results are shown
in Figure 5.5. Galacturonic acid is the major component of PS-II, PS-III, and PS-IV.
Substantial amounts of apiose are present in PS-II and PS-III but very little is present in PS-
IV, assuming those fractions analyzed were representative of all fractions constituting PS-IV.

- Small amounts of xylose, arabinose, and rhamnose were also detected in PS-II and PS-III

116

Figure 5.5. Sugar composition of the pectic polysaccharide(s) in individual column
fractions from the DEAE-Trisacryl Plus M column chromatography (Figure 5.3). The

profiles, total A520 ( O ) and salt concentration ( ----- -) are those of Figure 5.3. Sugars are:

galacturonic acid ( I ), apiose ( O ), xylose( V ), rhamnose ( O ) and arabinose ( E] ). Sugar

composition was determined as described in the Materials and Methods. The vertical dotted

lines and double-headed arrows have the same meaning as in Figure 5 .3.

117

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and a small amount of xylose was detected in PS-IV. The presence of these sugars was
confirmed by mass spectrometry. Gas chromatographic analysis also detected arabinose and
rhamnose in PS-IV and fucose, mannose, and glucose in all three polysaccharides, each at
less than 0.5 mole%, but their presence has not been confirmed by mass spectrometry. The
sugar composition across the samples constituting PS-IIb and PS—IVb was reasonably
constant (Figure 5 .5).

The polysaccharide material in 23 column fractions between fractions 207 and 301 of
Figure 5.3 was tested for galactose and none was detected. Approximately every third
fraction was analyzed except between fractions 231 and 284 where approximately every fifth
fraction was tested. Selective ion monitoring analysis showed that 100.4 :1: 0.8% (std. dev.)
of the galactitol peracetate, on a mole basis, was derived from the galacturonic acid residues
of the polysaccharide material and none from galactose residues. For column fractions 189
and 199, 91.3% and 96.7%, respectively, was derived from galacturonic acid residues and
8.7% and 3.3% from galactose residues.

Degree of methyl esterification. The degree of methyl esterification of the
polysaccharide material in column fractions 208, 215, 218, 222, 287, 289, 290, 292, 294 and
297 of Figure 5.3 ranged from 0 to 1.6% and averaged 0.25 :1: 0.56% (std. dev.).

’ H NMR spectroscopy. The low-field portion of the 500 MHz 1H NMR spectra for
F289, F290, F292, polygalacturonic acid and apple pectic acid were similar. The signals for
H—1, H-4 and H-5 were each a single symmetrical peak with no sign of splitting in the H-5
and H-1 signals. The 1H NMR spectra of F289 and polygalacturonic acid are shown in

' Figure 5.6. The spectrum of F223, a pectic polysaccharide containing a high amount of

119

Figure 5.6. The low-field region of the 500 MHz 1H N MR spectra of L. minor pectic
polysaccharide F289 (a) and polygalacturonic acid (b). The signals assigned to the
protons on C-1, C-4 and C-5 of galacturonosyl residues are labeled H-1, H—4 and H-5,

respectively.

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121

apiose and presumably small amounts of other neutral sugars, was too complex to determine
whether there was any splitting of the H-5 and H-1 signals (data not shown).
Rechromatography of pectic polysaccharides. Rechromatography of PS-IIb and PS-
IVb on DEAE-Trisacryl Plus M showed that each migrated as a single band that for PS—Ilb
was almost symmetrical and for PS-IVb was symmetrical (Figure 5.7). Recovery of PS-IIb
and PS-IVb from the columns, based on determination of uronic acid, was 96% and 92%,

respectively.

DISCUSSION

Procedures were developed for determining the homogeneity of plant cell wall pectic
polysaccharide material eluted from DEAE columns. The homogeneity of these
polysaccharides has not been determined previously. The use of MALLS together with
HPSEC for testing the homogeneity of the eluted polysaccharide material led to the
unexpected finding that four pectic polysaccharides are present in the 22°C ammonium
oxalate-soluble fraction from cell walls of L. minor. These results show there is not a
continuum in structure for pectic polysaccharides in this fraction.

Two major bands of pectic polysaccharide material, PS-IIb and PS-IVb, were eluted
from the DEAE column that were considered to be homogeneous with respect to sugar
composition and degree of methyl esterification; each, however, was different chemically.
The average sugar composition of the polysaccharide material in the column fractions

analyzed that constituted PS-IIb (Figure 5.3) was: GalA (54.5 :I: 3.2%) (std. dev.), Api (39.3

122

Figure 5.7. Column chromatograms of PS-IIb ( O ) and PS-IVb ( O ). The column was
DEAE-Trisacryl Plus M. The two samples were run on separate but identical columns and
the columns were developed identically. Sample preparation, column operation, and

analyses were performed as described in the Materials and Methods.

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:1: 1.4%), Xyl (2.5 i 1.4%), Ara (1.4 :t 0.8%), and Rha (2.3 :t 0.3%). The average sugar
composition of the polysaccharide material in the column fractions analyzed that constituted
PS-IVb (Figure 5.3) was: GalA (96.3 i 0.8%), Api (2.1 :1: 0.7%), and Xyl (1.6 i 0.3%). The
inclusion of xylose, arabinose, and rhamnose as constituents of PS-IIb and xylose as a
constituent of PS-IVb is tentative since data establishing them as structural components of
the polysaccharides have not yet been obtained. In addition, small amounts of fucose,
mannose, and glucose and arabinose, rhamnose, fucose, mannose, and glucose were
tentatively detected in PS-IIb and PS-IVb, respectively. Whether these minor sugars are
constituents of the polysaccharides is also not known. The degree of methyl esterification
of the pectic polysaccharide material in the column fractions constituting both PS-IIb and PS-
IVb was basically zero. The 1H NMR results are in agreement with the above results. The
lack of detectable splitting in the H—5 and H-1 signals of F289, F290, and F292, the similarity
of their spectra to those of polygalacturonic acid and apple pectic acid and the lack of signals
for the other sugars also indicate the degree of methyl esterification and the neutral sugar
content of PS-IVb are low. The lower limit of detection of methyl esterification with a 400
MHz instrument is approximately 8% (18). The spectrum of F223 indicates that one or more
sugars, in addition to galacturonic acid, are present in PS-IIb.

PS-IIb and PS-IVb are heterogeneous with respect to size of molecules. The peak MW
of the polysaccharide molecules constituting PS-IIb ranged from 28, 100 - 97 ,000 while those
constituting PS-IVb ranged from 53,300 to at least 163,000. The M., range of cell wall pectic
polysaccharides from other plants determined by an absolute method such as MALLS has

not been reported. Light scattering was used to determine that the M., of a commercial sugar

125
beet pectin is 266,000 (19). The other light scattering studies on pectic polysaccharides were
done with material from fruit tissue (19-23).

Pectic polysaccharide samples in individual column fractions, before being combined
as part of PS-Ilb and PS-IVb, each migrated as a single band on HPSEC. The combined
samples, PS-IIb and PS-IVb, also each migrated as a single symmetrical or almost
symmetrical peak when chromatographed on ion-exchange (Figure 5.7) and HPSEC (data
not shown) columns. These results showed that PS-IIb and PS-IVb were free of
contaminants that migrated differently on these columns. This indicated further that they
were highly purified.

A pectic polysaccharide like PS-IVb, that is, one of high galacturonic acid content (2 96
mole %) and low degree of methyl esterification (~0%) and isolated under mild conditions,
has not been reported previously. The isolation of homogalacturonans has been reported but
the strong isolation conditions suggest these were derived from parent polysaccharides by
degradation during the isolation process (24-26). The degree of methyl esterification of these
isolated homogalacturonans was not reported. A pectic polysaccharide of high galacturonic
acid content (94 mole %) was isolated from Zea mays L. shoots under relatively mild
conditions but the degree of methyl esterification was also not reported (27). Pectic
polysaccharides of high galacturonic acid content (2 90 mole%) have been isolated from
various fruit tissues but all have either a high degree of methyl esterification (2 44 %) or
were isolated from cell fractions with a high degree of methyl esterification (2 54 %) (8,28-
30). PS-IVb from L. minor was discovered only after conditions were found that gave

quantitative recovery of pectic polysaccharides from the DEAE-Trisacryl Plus M column

126

(15). This type of pectic polysaccharide is probably present in other plants but has escaped
detection because of its tight binding to anion-exchange columns.

In the procedure developed for isolating pectic polysaccharides, all steps that involved
concentration, dialysis, or freeze—drying of polysaccharide solutions were eliminated as were
those that involved precipitation of polysaccharides from solution. The cell walls were
isolated from fresh plants at 04°C and pH 5.5, the extraction time with ammonium oxalate
was short, 30 min (2), and the extraction was performed at 22°C. The extract was applied
directly to the DEAE column and the column buffer was the same as the extracting agent
(0.05 M ammonium oxalate). The recovery of pectic polysaccharides from the DEAE column
was quantitative and the column was operated at pH 5.5. The procedures and conditions
used for isolation of the pectic polysaccharides eliminated losses and minimized degradation
reactions of the polysaccharides.

Three general types of pectic polysaccharides have been reported to be present in plant
cell walls. They are commonly designated RG-I, RG-II, and homogalacturonan. Our results
show four pectic polysaccharides are present in the 22°C ammonium oxalate-soluble fraction
from cell walls of L. minor. Based on our structural results, PS-IV would be classified as
a homogalacturonan. PS-II contains rhamnose but the rhamnose residues are linked
terminally and are not within the backbone (Chapter VI), which means PS-II can not be
classified as a RG-I or RG-IL Pectic polysaccharides containing rhamnosyl residues only as
terminal groups have not been reported. There is not sufficient homogeneity and structural
information to classify PS-I and PS-III. As stated earlier, the light scattering results show

there is not a continuum in structure between these four pectic polysaccharides.

127

A simple, mild procedure for quantitatively isolating pectic polysaccharides from the
22°C ammonium oxalate-soluble fraction prepared from purified cell walls of L. minor was
developed. Four different pectic polysaccharides were isolated. Procedures for determining
the homogeneity of the isolated pectic polysaccharides were developed. Two of the pectic
polysaccharide fractions isolated, PS-IIb and PS-IVb, were chemically homogeneous with
respect to sugar composition and degree of methyl esterification but were heterogeneous with

respect to the size of their molecules.

(1)
(2)
(3)

(4)
(5)
(6)

(7)
(8)

(9)
(10)

(11)

(12)

(13)

(14)
(15)
(16)

128

REFERENCES

Carpita, N. C. and Gibeaut, D. M. (1993) Plant J., 3, 1-30.
Kindel, P. K., Cheng, L. and Ade, B. R. (1996) Phytochemistry, 41, 719-723.
Souty, M. , Thibault, J. -F., Navarro-Garcia, G., Lopez-Roca, J. -M. and Breuils, L.
(1981) Sciences des Aliments, 1, 67-80.
Redgwell, R. J. and Selvendran, R. R. (1986) Carbohydr. Res., 157, 183-199.
Saulnier, L. and Thibault, J. -F. (1987) Carbohydr. Polymers, 7, 329-343.
Renard, C. M. G. C.,Voragen, A. G. J .,Thibault, J. -F. and Pilnik, W. (1990)
Carbohydr. Polymers, 12, 9-25.
Ryden, P. and Selvendran, R. R. (1990) Carbohydr. Res., 195, 257-272.
Seymour, G. B., Colquhoun, I. J ., DuPont, M. S., Parsley, KR. and Selvendran, R. R.
(1990) Photochemistry, 29, 725-731.
Ryden, P. and Selvendran, R. R. (1990) Biochem. J., 269, 393-402.
Coimbra, M. A., Waldron, K. W. and Selvendran, R. R. (1994) Carbohydr. Res.,
252, 245-262.
Gooneratne, J ., Needs, P. W., Ryden, P. and Selvendran, R. R. (1994) Carbohydr.
Res., 265 61-77.
Kindel, P. K. and Watson, R. R. (1973) Biochem. J., 133, 227-241.
DuBois, M., Gilles, K. A., Hamilton, J. K., Roberts, P. A. and Smith, F. (1956) Anal.
Chem, 28, 350-356.
Blumenkratz, N. and Asboe-Hansen, G. (1973) Anal. Biochem., 54, 484-489.
Cheng, L. and Kindel, P. K. (1995) Anal. Biochem., 228, 109-114.

Kruis, A. (1936) Z. physik. Chem. (B), 34, 13-50.

(17)
(18)

(19)

(20)

(21)

<22)
(23)
(24)
(25)
(26)
(27)

(28)

(29)

(30)

129

P. J. Wood and I. R. Siddiqui, (1971) Anal. Biochem., 39, 418428.

Grasdalen, H., Bakoy, O. E. and Larsen, B. (1988) Carbohydr. Res., 184, 183-191.
Thibault, J. -F., Renard, C. M. G. C., Axelos, M. A. V., Roger, P. and Crépau, M. -J.
(1993) Carbohydr. Res., 238, 271-286.

Chapman, H. D., Morris, V. J ., Selvendran, R. R. and O’Neill, M. A. (1987)
Carbohydr. Res., 165, 53-68.

Kontominas, M. G. and Kokini, J. L. (1990) Lebensm-Wiss. u. Technol, 23, 174-
177.

Smith, J. E. and Stainsby, G. (1977) British Polymer J., 9, 284-289.

Doco, T. and Brillouet, J. -M. (1993) Carbohydr. Res., 243, 333-343.
Bhattacharjee, S. S. and Timell, T. E. (1965) Can. J. Chem, 43, 758-765.

Zitko, V. and Bishop, C. T. (1965) Can. J. Chem, 43, 3206-3214.

Chambat, G. and Joseleau, J. —P. (1980) Carbohydr. Res., 85, C10-C12.

Kato, Y. and Nevins, D. J., (1989) Plant Physiol., 89, 792-797.

Vries, J. A. De, Voragen, A. G. J., Rombouts, F. M. and Pilnik, W. (1981)
Carbohydr. Polymers, 1, 117-127.

Redgwell, R. J ., Melton, L. D. and Brasch, DJ. (1988) Carbohydr. Res., 182, 241-
258.

Westerlund, B., Aman, P., Andersson, R. and Andersson, R. E. (1991) Carbohydr.

Polymers, 15, 67-78.

CHAPTER VI

STRUCTURAL ANALYSIS OF PECTIC POLYSACCHARIDES

PS-IIb AND PS-IVb

130

131

ABSTRACT

Methylation analysis and partial hydrolysis followed by poly-[1,4-a-D-galacturonide]
glycanohydrolase (EC 3.2.1.15) digestion were performed on PS-IIb and PS-IVb, two pectic
polysaccharides purified from cell walls of L. minor. Glycosyl-linkage composition of the
two was deduced from the electron impact mass spectrometry of partially 0-methylated
alditol acetates derivatives. With 97% galacturonosyl residues (4-1inked:terminal:2,4-linked
:3,4-linked residues = 92.1:1.9:1.7:1.3) and only 0.5%, 1.0% and 1.6%, respectively, of
terminal rhamnosyl, terminal apiosyl and internal apiosyl residues, PS-IVb is virtually a
homogalacuronan. In contrast, PS-IIb is highly branched with side chains mainly of 1,3'-
linked apiobiose (the mole% of teminal apiose and internal apiose is 21.3% and 27.7%,
respectively) attached almost solely at the 2-0-position of every other galacturonosyl
residues of the backbone. Direct digestion of these two polysaccharides with poly-[1,4-a-D-
galacturonide] glycanohydrolase released 98.5% of the galacturonosyl residues of PS-IVb
while only 12% of the galacturonosyl residues of PS-Ilb were released. However, after the
side chains were removed from PS-Ilb by partial acid hydrolysis, the enzyme digestion
released all the galacturonosyl residues. The results indicate both PS-IIb and PS-IVb have
a backbone of a- 1 ,4-linked-D-galacturonosyl residues. PS-IIb but not PS-IVb has side chains
of apiose and apiobiose. The side chains of PS-IIb cover most of the backbone and occur on

approximately every the other galacturonosyl residue.

132

INTRODUCTION

Pectic polysaccharides are usually the most abundant component of plant primary cell
wall. The major types of pectic polysaccharide fractions isolated from plant cell walls are
homogalacturonan (1-5), RG-I (6-11) and RG-II (12-17). However, no pure
homogalacturonan with zero degree of methyl esterification has been isolated from primary
cell walls without using treatments that are likely to cleave covalent bonds. The rhamnosyl
residues in RG-I are 1,2-linked and approximately half of them are branched, containing
glycosyl substitutes at 0—4 (3). A large portion of the rhamnosyl residues of RG-II are 3-,
3,4-, 2,3,4- and terminally linked.

Pectic polysaccharides solubilized from the cell wall of L minor with 0.5% ammonium
oxalate have been found to be different from the above three types; they contain mainly
galacturonosyl and apiosyl residues and were partially characterized as apiogalacturonans
(1820). Beck (18) detected that apiose (25.2-27.9% of the apiogalacturonans), xylose
(8.3 96), galactose (2.8%) and apiose-free oligo- and mono galacturonic acid by paper
chromatography after mild acidic hydrolysis, autohydrolysis and pectinase (a mixture of
exo— and endo-polygalacturonase) digestion of the apiogalacturonans. Based on these
results Beck suggested that the apiogalacturonan he isolated was "an unesterified rat-1,4
linked polygalacturonic acid to which monomeric side-groups (75 % apiose and 25 % xylose
residues) are attached" (18). The apiogalacturonan fraction isolated and purified from cell
walls of L. minor by Hart and Kindel (19,20) consisted mainly of D-galacturonosyl and

apiosyl residues (apiose accounted for 7.9-38. l % of the apiogalacturonans). Mild acid

133

hydrolysis of the apiogalacturonans gave three products: apiose, apiobiose and
galacturonan (20). The galacturonan was hydrolyzed with a crude pectinase, providing
some evidence that the galacturonosyl residues were or-l ,4 linked. Periodate oxidation
together with proton NMR analysis suggested that apiobiose had the structure, B-D—Apif-
(1-+3')-D-Api. However, complete methylation analysis was not performed by either
Beck’s or Kindel's group. It was not established whether the apiosyl and apiobiosyl
residues were linked to 02 or C-3 of the residues of the galacturonan backbone. In
addition, the homogeneity of the apiogalacturonans had not been determined when the
polysaccharides were partially characterized previously. With current techniques such as
GC-MS and HPSEC-MALLS, we [Cheng and Kindel (21), Chapter V] re-examined the
isolation and purification procedures and have purified two chemically different pectic
polysaccharides, PS-IIb and PS-IVb, from cell walls of L. minor. With a peak M“, range
of 28, 100 -99,700, PS-IIb consisted mainly of galacturonosyl (54.5;t3.2%) and apiosyl
(39.31: 1.4%) residues although small amounts of rhamnosyl (2.3130396), xylosyl
(2.51 1.4%) and arabinosyl (1.4 i0.8%) residues have also been detected with GC-MS.
PS-IVb is virtually a homogalacturonan since 96.3% (10.8) of the polysaccharide is
galacturonosyl residues. Api, 2.1i0.7%, Xyl, 1.6 i0.3% and other neutral sugars, <
0.5% were also found. PS-IVb has a peak M., range of 53,300 to at least 163,000. In this
study, methylation analysis, partial hydrolysis and polygalacturonase digestion followed by

HPAEC-PAD were performed on these two pectic polysaccharides.

134

MATERIALS AND METHODS

General method. Total carbohydrate and uronic acids were measured with the
methods of Dubois et al. (22) and Blumenkrantz and Asboe-Hansen (23), respectively.

Materials. Sodium borohydn'de, sodium borodeuteride (98% D), anhydrous dimethyl
sulfoxide, methyl iodide were obtained from Aldrich. l-Ethyl-3-(3-dimethylaminopropyl)
carbodiimide, pectinase (purified, poly-[1,4-a-D-galacturonide]glycanohydrolase, EC
3.2.1.15) and bovine serum albumin was obtained from Sigma. DEAE-Trisacryl Plus M
column fractions 195-222 and 287-327 (Chapter V, Figure 5.3), 4 mL each fraction, were
combined as PS-IIb and PS-IVb, respectively. The samples were dialyzed against distilled
water for 45 h with 10 water changes and tested for uronic acid and total sugar.

Carboxyl reduction. The dialyzed samples PS-IIb and PS-IVb were evaporated to ~10
mL under 40°C and the carboxyl groups reduced basically as described by Taylor and
Conrad (24) except that after 173 mg of EDAC was added, 0.01 M and 0.05 M HCl was
added dropwise by hand to maintain the pH at 4.75 for the 2 h, and 0.5 M and 5 M HCl was
added dropwise to maintain the pH at 7.0 while about 25 mL of 2 M NaBH4 was added over
a 2 h period. The samples were dialyzed in tubing with a 6000-8000 molecular weight cut-
off against distilled water for 36 h and evaporated under 40°C to about 10 mL. The
carboxyl-reduction procedure was repeated once. Uronic acid and total sugar were
determined for the dialyzed samples and the above procedure of carboxyl reduction was
repeated once for PS-IVb. The final volume of PS-IVb after evaporation was 15.9 mL.

Methylation. Samples of carboxyl-reduced PS-IIb, 2 mL (0.826 mg/mL by total sugar

135

and uronic acid tests), and PS-IVb, 3.7 mL (0.439 mg/mL by total suagr and uronic acid
tests), were lyophilized to dryness in 1 DRAM vials and dried in vacuo over P205 for 2 days.
The polysacharides were methylated by a modification of the procedure of Ciucanu and
Kerek (25) as described by Needs and Selvendran (26) except the following changes were
made: Each samples was dissolved in 1 mL of anhydrous DMSO under N2 , sonicated for
5 min and stirred slowly overnight. After 100 mg of fine powdered anhydrous NaOH was
added (under N2), the sample was briefly vortexed, sonicated for 20 min, stirred for 1 h and
the sonication and stirring were repeated once with the last stirring being 2 h. Methyl iodide,
0.8 mL, was added dropwise over 2 min to the sample which was held by a pre-cooled (in
freezer for 15 min) metal block while being stirred under N2. The mixture was briefly
vortexed, sonicated for 15 min and stirred for 30 min. Methyl iodide, 0.25 mL, was added
and the sample was vortexed, sonicated and stirred as above. The mixture was transfered to
2 DRAM vials, 3 mL of water was added and the solution was extracted with chloroform
three times each time with 1.5 mL. The chloroform phase was washed with water (3 x 3
mL) and reduced to 1 mL with N2. The water phase was diluted to 15 mL and slowly applied
to a Sep-Pak C18 Cartridge (Waters Corp., Milford), which was pre-treated as described (27).
The cartridge was washed with 20 mL water and methylated polysaccharides were eluted
with acetonitrile and ethanol as described (27). The effluent was reduced to 1 mL with N2,
combined with the chloroform phase and the solution taken to dryness.

Hydrolysis, reduction and acetylation. Per-O-methylated PS-IVb was treated with 2
M HCl in dry methanol at 80°C for 4 h and the sample was dried with N2 at 22°C.

Methylated PS-Ilb and methanolysis-treated, methylated PS-IVb were hydrolyzed with 0.5

136

mL of 2 M TFA at 120°C for 90 min and the samples were dried with N2 at 22°C. NaBD4,
2.5 mg in 0.25 mL of 5% (v/v) 14 M ammonium hydroxide in ethanol, was added and the
samples were vortexed and stood at 22°C for 3 h. Acetic acid, 25 aL, was added and the
samples were brought just to dryness with N2 at 22°C . Acetic acid-methanol (1 :9, v/v), 0.5
mL, was added to the samples and they were dried with N2 at 22°C. The addition and
evaporation of 1:9 (v/v) acetic acid-methanol was repeated three times and followed by two
cycles of evaporations to dryness with 1 mL of methanol. Pyridine-acetic anhydride (2:1,
v/v), 0.25 mL, was added and the samples were heated at 80°C for 45 min. After the vials
were cooled to 22°C, 2 mL of water was added and the mixture was extracted with
chloroform (3 x 1 mL). The chloroform extract was reduced to 1.5 mL with N2 and washed
with water (3 x 3 mL). The PMAAs in the chloroform phase were transferred to a 0.5
DRAM vial, the solution was reduced to ~0.15 mL with N2 and analyzed.

GC-MS determination of the partial methylated alditol acetates. Separation and
identification of PMAAs were by GC (FID) and GC-MS basically as was done with alditol
acetates (28), except that a 30 m DB-225 capillary column (0.25 m, i. d., for GC and
0.32 m, i. d., for GC-MS) was used. For GC the temperature was started at 180°C and
increased at a rate of 2.5°C/min to 230°C. For GC-MS the initial temperature was
100°C, and was increased at a rate of 45°C/min to 170°C then increased at a rate of
2.5°C/min to 225°C. The molar ratio of the PMAAs was determined from the individual
GC peaks by using the molar response factors calculated on the basis of effective carbon
response (e.c.r) as described (29-31).

Pectinase degradation of polysaccharides PS-IIb and PS-IVb. Pectinase (1.1 units/

137

mg) was dissolved in aq. 0.5% (w/v) BSA containing 50 mM sodium acetate buffer, pH 5.0,
at 10 mg/mL. One tenth (0.1) mL of a solution containing either polysaccharide PS-IIb or
PS-IVb (1.2 mg/mL, uronic acid and total sugar tests) was diluted with 0.1 mL of 0.1 M
sodium acetate buffer, pH 5.0. Pectinase solution or BSA [aq. 0.5% (w/v) solution
containing 50 mM sodium acetate, pH 5.0], 10 al., was added and the mixture was incubated
at 30°C for 4 h. Samples of polysaccharides PS-Ilb and PS-IVb were also partially
hydrolyzed at pH 4 and 100°C for 5 h before treatment with pectinase. The partially
hydrolyzed PS-IIb and PS-IVb were digested with pectinase as described above.

High performance anion exchange chromatography with pulse amperometric detection
PS-IIb and PS-IV b samples treated with pectinase, partial acid hydrolysis or both, plus
controls were diluted 1:3 with water and analyzed by HPAEC-PAD (Dionex system). The
Dionex system was equipped with a CarboPac PAl coltunn (4 mm X 250 mm) and a
CarboPac PAl guard column and was operated at 22°C. Samples of 20 pL were injected
and the column flow rate was 1 mL/min. The following eluents were used: 0-18 min, 20
mM NaOH; 18-45 min, concentration of NaOH was increased to 100 mM and sodium
acetate was increased from 0 to 1 M, each linearly; 45—55 min, the concentration of NaOH
and sodium acetate was kept unchanged; 55—60 min, concentration of NaOH was decreased
from 100 mM to 20 mM and that of sodium acetate was decreased to 0 M, each linearly.
Chromatograms were recorded and integrated with a HP3390 integrator and also with a
computer that was connected to the Dionex system through a SRI interface and operated
with PEAK-II software (SRI Instruments, Torrance, California). Standard samples of

galactose, apiobiose, and galacturonic acid were used to calibrate and quantify the

138

HPAEC-PAD system. The percentage of free GalA released in samples was calculated
from GalA detected by HPAEC-PAD and from results of total carbohydrate (22) and

uronic acid (23) tests of the original polysaccharides (Appendices C and D).

RESULTS

Methylation analysis. Gas chromatograms of PMAAs from methylation analysis
of PS-llb and PS-IVb are shown in Figure 6.1. The 11 PMAAs were identified by GC-MS
analysis. The GC-MS spectra of PMAAs 1, 3-5, and 7-11 are similar to those given by
Carpita and Shea (32). PMAAs of apiose were not described in ref. 32 or elsewhere in the
literature. Therefore spectra for l,4-di-0-acetyl-(1-deuterio-)-2,3 ,3 '-tri-0-methyl apiitol
(t-Apif, the glycosidic linkage at C-1 is assumed for all PMAA in this study) and 1,3' ,4-tri-
0-acetyl-(1-deuterio-)-2,3-di-0-methyl apiitol (3'-Apif) are not available. Peak 1 (Figure
6.1a), l,5—di-0-acetyl-(1-deuterio-)-2,3,4—tri-0-methyl-6—deoxy-hexitol was assigned to t-
Rhap since sugar composition analysis (Chapter V) showed that no significant amount of
any other 6—deoxysugar (such as fucose) was present. Peak 1 of Figure 6. lb had a similar
GC retention time and mass spectra (data not shown) as peak 1 of Figure 6.1a and
therefore was considered to be t-Rhap although sugar composition analysis did not
conclusively detect Rha in PS-IVb possibly due to the small quantity present. The
following information was used to identify peaks 4, 8, 10 and 11 of Figures 6.13 and b.
First, the mass spectrum of each peak was similar to corresponding hexose PMAA

described by Carpita and Shea (32). Second, enzyme degradation indicated that

139

Figure 6.1. Gas-liquid chromatograms of partial methylated alditol acetates derived
from pectic polysaccharides PS-IIb (a) and PS-IVb (b). Experimental procedures are
described in the Materials and Methods. For both panels, peaks 1 through 12 are: [1], 1,5-di-
0—acetyl-(1-deuterio-)-2,3,4-tri-0-methyl-6-deoxy-hexitol (t-Rhap); [2], l ,4-di-0-acetyl-(1-
deuterio-)-2,3,3'-tri-0-methyl apiitol (t-Apif); [3], 1,5-di-0-acetyl-(1-deuterio-)-2,3,4-tri-0-
methyl pentitol (t-pentitolp); [4], 1,5-di-0-acetyl-(l-deuterio-)-2,3,4,6-tetra-0-methyl
hexitol (t-GaIpA); [5], 1,4,5-tri-0-acetyl-(1-deuterio-)-2,3-di-0-methyl pentitol (4-pentitolp,
or S-pentitolf); [6], 1,3',4-tri-0-acetyl-(1-deuterio-)-2,3-di-0—methyl apiitol (3'-Apij); [7],
1,4,5-tri-0-acetyl-(1-deuterio-)-2,3,6-tri-0-methyl hexitol (4-hexitolp, or S-hexitolt); [8],
1 ,4,5-tri-0—acetyl-(1-deuterio-)-2,3,6-tri-0-methyl hexitol (4-Ga1pA); [9], 1,4,5-tri-0-acetyl-
(1-deuterio-)-2,3,6-tri-0—methyl hexitol (4-hexitolp, or S-hexitolf); [10], 1,3,4,5-tetra-0-
acetyl-(l-deuterio-)-2,6-di—0-methyl hexitol (3,4-GalpA); [11], 1,2,4,5-tetra-0—acetyl-(1-
deuterio-)-3,6-di-0-methyl hexitol (2,4-GalpA); [12], myo-inositol hexaacetate (internal

standard).

140

 

 

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141

galacturonosyl residues were in the pyranose form. Third, hexoses other than GalpA were
not conclusively identified by sugar composition analysis (Chapter V). Although peaks
from GC with retention times the same as those of mannose and glucose were detected,
they were present in a trace amount (< 0.5 %) compared to the large amount of 031A in
PS-IVb (96.3%) and PS-Ilb (54.5%). Since the area ratio of peaks 7, 8, and 9 (PMAAs
of hexoses) in Figure 6.1a (PS-Hb) is 1:30:l and in Figure 6.1b (PS-IVb) it is 1:50;] and
assuming the major PMAA peak of hexoses was derived from GalA, then peak 8 can only
be assigned to 4-GalpA. The same reasoning was used to identify peak 11 of Figure 6.1a
as 2,4-GalpA. The other two hexose peaks, peaks 7 and 9 have to be the PMAAs derived
from Man and Glc residues although the data available do not differentiate between the
two. Since the PMAAs of Man and Glc were assigned to PMAA peaks 7 and 9 (Figure
6.1), the terminal and 3,4- linked hexoses (peaks 4 and 10, Figure 6.1) were tentatively
identified as t-GalpA and 3 ,4- linked GalpA, respectively, although standard samples are
necessary for positive identification.

The identification of t—Apif and 3'-Apif in PS-lIb was based on: (i) the mass spectra of
the PMAAs and the fragmentations proposed in Figures 6.2 and 6.3, respectively, (ii) the
high amount of Api in PS-Ilb as determined by sugar composition analysis (Chapter V) and
(iii) the earlier isolation of apiobiose from pectic polysaccharides of L minor (20). Ions at
m/z 101, 146, 161, 205 and 206 (Figure 6.2) are characteristic of t-Apif and ions at m/z 129
and 189 (Figure 6.3) are characteristic of 3'-Apif. Sugar composition results (Chapter V)
showed that the Api content of PS-IVb is low (2.1:t0.7%). However, the similarity of

retention times and mass spectra of two PMAAs from PS-IVb with those of t-Apif and 3'-

142

Figure 6.2. E.i. mass spectrum and proposed fragmentation of 1,4-di-0-acetyl-(l-

deuterio-)-2,3,3'-tri-0-methyl apiitol (t-Api, peak 2 of Figure 6.1a).

143

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144

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145

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146
Apif from PS-Hb plus criterium (iii) above, identify t-Apif and 3'-Apif as components of
PS-IVb. The glycosyl-linkage compositions of PS-IIb and PS-IVb, calculated by using
effective carbon response, are shown in Table 6.1.

Enzyme degradation of polysaccharides PS-Ilb and PS-I Vb. HPAEC-PAD
chromatograms of PS-IIb, pectinase-digested PS-Ilb, partially acid hydrolyzed (pH 4.0,
100°C, 5 h) PS-IIb, and PS-Ilb partially acid hydrolyzed and then digested with pectinase are
shown in Figures 6.4a-d. HPAEC-PAD chromatograms of PS-IVb subjected to the same
four treatments are shown in Figures 6.5a-d. Free galacturonic acid was not released in the
controls (Figures 6.4a and 6.5a). The amount of GalA released by treatment of lPS-IIb and
PS-IVb with pectinase alone was 12% (peak 4, Figure 6.4b) and 99% (peak 4, Figure 6.5b),
respectively, of the GalA found in the starting polysaccharides. Partial acid hydrolysis of PS-
IIb at pH 4.0 and 100°C for 5 h appeared to have released most of the apiose and apiobiose
residues (compare peaks 2 and 3, respectively, of Figure 6.4c to those of Figure 6.4d) and
only 2.2% of the GalA (peak 4, Figure 6.4c) from the polysaccharide; however, good
quantification of apiose and apiobiose was not established due to irregular elution of these
compounds with this system. PS-IVb, a virtual homogalacuronan, was quite resistant to acid
hydrolysis at pH 4.0 and 100°C for 5 h since only 1.1% of the GalA (peak 4, Figure 6.5c)
was released form the polysaccharide. The GalA released from PS-IIb and PS-IVb by partial
acid hydrolysis followed by digestion with pectinase was 106% (peak 4, Figure 6.4d and

65d) of the GalA found in the starting polysachharides.

147

Table 6.1. Glycosyl-linkage composition (mol%) of PS-IIb and PS-IVb from

methylation analysis.

 

 

 

Peak number Glycosyl Polysaccharide

in Figure 6.1 linkage PS-IIb PS-IVb
l t-Rhap 3.30 0.48
2 t-Apif 21 .30 1 .02
6 3'-Apif 27.65 1.55
4 t-GalpA 2.1 1 1.87
8 4-GalpA 23.34 92.05
10 3,4-GalpA 0.90 1 .30
l 1 2,4-GalpA 21.40 1 .73

 

148

Figure 6.4. Column chromatograms from HPAEC-PAD for polysaccharide PS-IIb
after various treatments. The chromatograms shown are : (a), PS-Hb (as control); (b), PS-
IIb digested with poly-[1,4-a-D-galacturonide] glycanohydrolase; (c), PS-IIb hydrolyzed at
pH 4.0 and 100°C for 5 h; (d), PS-IIb digested with poly-[1,4-a-D-galacturonide]
glycanohydrolase after hydrolysis at pH 4.0 and 100°C for 5 h. Peaks 1 to 4 are Gal (internal
standard), Api, apiobiose and GalA, respectively. Oligouronic acids with a degree of

polymerization greater than 15 can not be detected with this system.

 

 

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Figure 6.5. Column chromatograms from HPAEC-PAD for polysaccharide PS-IVb
after various treatments. The chromatograms shown are : (a), PS-IVb (as control); (b), PS-
IVb digested with poly-[1,4-or-D-galacturonide] glycanohydrolase; (c), PS-IVb hydrolyzed
at pH 4.0 and 100°C for 5 h; (d), PS-IVb digested with poly-[1,4-a-D-galacturonide]
glycanohydrolase after hydrolysis at pH 4.0 and 100°C for 5 h. Peaks 1 to 4 are Gal (internal
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DISCUSSION

For PS-Ilb, the ratio, PMAAs of apiosyl residueszPMAAs of galacturonosyl residues
(49%:48%), does not exactly match but is reasonably close to the ratio, ApizGalA
(40%:55%) obtained from sugar composition analysis by GC of alditol acetates. PS-IIb
clearly is a highly branched pectic polysaccharide, since the glycosyl-linkage composition
(Table 6.1) shows that nearly half (22% out of 48%) of its galacturonosyl residues are
branched. Almost all (96%) of the branching is at C-2 of the galacturonosyl residues.
Consequently it is not surprising to find that PS-Ilb is quite resistant to poly-[1,4-a-D-
galacturonide] glycanohydrolase digestion; only 12% of the galacturonosyl residues were
released by the enzyme treatment.

The following results indicate that most of the apiose in PS-lIb is present as apiobiosyl
side chains that are attached to galacturonosyl residues: (i) the amount of terminal apiose is
close to that of internal apiose (t-Apifi 3'-Apif =21.3%:27.7%); (ii) partial acid hydrolysis
of PS-IIb at 100°C appeared to have released most of the apiobiose (compare peaks 2 and
3 of Figure 6.4c to those of Figure 6.4d), but no significant amount of GalA was released;
(iii) after partial acid hydrolysis, all galacturonosyl residues were released as free GalA from
PS-IIb by treatment with poly-[1,4-a-D-galacturonide] glycanohydrolase. These findings are
in agreement with earlier findings (19,20).

The complete release of galacturonosy residues as free GalA from PS-IIb by poly-[140:-
D-galacturonide] glycanohydrolase after mild acid hydrolysis indicates that the polysaccharide

has a backbone of a-1,4-linked D-galacturonic acid. Since: (i) the ratio of branched

153

galacturonosyl residues (2,4-Ga1pA plus 3,4-GalpA) to unbranched residues (4-Ga1pA) was
approximately 1:1 (22%:23%), (ii) the ratio of PMAA of galacturonosyl residues (t-GalpA,
4-GalpA, 3,4—GalpA and 2,4-GalpA) to those of apiosyl residues (t-Apif and 3'—Apif ) was
also approximately 1:1 (48%:49%), and (iii) untreated PS-IIb was quite resistant to the
enzyme digestion, the apiobiose side chains must be relatively evenly distributed along the
backbone on approximately every other galacturonosyl residue.

Since: (i) internal apiose (3'-Apif, 27.7%) was somewhat higher than the terminal apiose
(21.3%), (ii) rhamnosyl residues (3.3%) were solely terminal-linked, and (iii) the release of
free GalA from the backbone was complete after side chains were removed by partial
hydrolysis, it is possible that rhamnosyl residues may be attached to the backbone of the
polysaccharide through apiose. However, evidence for a disaccharide of Rha and Api has
not been obtained.

Although small amounts of 1,5-di-0-acetyl-(l-deuterio-)-2,3,4-tri-0-methyl pentitol,
l ,4,5-tri-0-acetyl-( l-deuterio-)-2, 3-di-0-methyl pentitol and 1,4,5-tri-0-acetyl-(1-deuterio-
)-2,3,6-tri—0-methyl hexitol were found by methylation analysis and le (2.5 :1; 1.4 96), Ara
(1.4 10.8%) and trace amounts (<0.5%) of other hexose, such as Man and Glc, were
found by sugar composition analysis, the assignment of the corresponding sugar to these
PMAAs has not been possible due to lack of standards. However the finding that complete
degradation of PS-llb by pectinase occurred after partial acid hydrolysis seemed to indicate
that these minor components (assuming they are not contaminants) are in side chains that are
released by the acid hydrolysis.

All PMAAs obtained from PS-llb (Figure 6.1a) were also obtained from PS-IVb (Figure

154

6.1b). The major difference between PS-IVb and PS-IIb is that in PS-lVb the PMAAs
obtained in large amounts were all derived from galacturonosyl residues while in PS-IIb they
were derived from both galacturonosyl and apiosyl residues. Although small amounts of
PMAAs of neutral sugars were detected from methylation analysis of PS-IVb, and Api
(2.1%), Xyl (1.6%) and trace amounts (<0.5%) of other neutral sugars were detected by sugar
composition analysis (Chapter V), these sugars may actually be present in PS-III and PS-III
is contaminating PS-IVb. The overlapping elution of PS-III and PS-IV from the DEAE
column (Chapter V) makes this a possiblity. The molar percentage of PMAAs of PS-lVb
detected by methylation analysis (97%, t-GalpA, 4—GalpA, 3,4-GalpA and 2,4-GalpA) is
similar to the molar percentage of GalA detected in sugar composition analysis (Chapter V).
The high content of GalA and the fact that PS-IVb was nearly completely digested by

pectinase indicate PS-IVb is a homogalacturonan.

10.

11.

12.

l3.

I4.

155

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Ishii, S. (1982). Phytochemistry 21, 778-780.

Beck, E. (1967). Z. Pflanzenphysiol. Bd. 57, S. 444-461

Hart, D. A. and Kindel, P. K. (1970) Biochem. J., 116, 569-579

Hart, D. A. and Kindel, P. K. (1970) Biochemistry 9, 2190-2196

Cheng, L. and Kindel, P. K. (1996) Homogeneity and Structure of the 22 °C Ammonium
Oxalate-soluble Pectic Polysaccharides of Lemna minor, submitted to Carbohydr. Res.
M. G. Dubois, K. A. Gilles, J. K. Hamilton, P. A. Rebers and F. Smith (1956) Anal.
Chem, 28, 350-356.

N. Blumenkrantz, and G. Asboe-Hansen (1973) Anal. Biochem., 54, 484-489

R. L. Taylor and H. E. Conrad (1972) Biochemistry, 11, 1383-1388.

I. O. Ciucanu and F. Kerek (1984) Carbohydr. Res., 131, 209-217.

P. W. Needs and R. R. Selvendran (1993) Carbohydr. Res., 245, 1-10.

T. J. Waeghe, A. G. Darvill, M. McNeil and P. Albersheim (1983) Carbohydr. Res.,
123, 281-304.

P. K. Kindel, and L. Cheng (1990) Carbohydr. Res.,l99, 55-65.

D. P. Sweet, R. H. Shapiro, and P. Albersheim (1975) Carbohydr. Res., 40, 217-225.
R. F. Addison and R. G. Ackman (1968) J. Gas Chromatogr., 6, 135-138.

R. G. Ackman (1964) J. Gas Chromatogr., 2, 173-197.

157
32. Carpita, N. C. and Shea, E. M. Analysis of Carbohydrates by GLC and MS (Biermann,

C. J. and McGinnis, G. D., Eds). CRC Press, 1990, Chapter 9, 157-216.

CHAPTER VII

CONCLUSIONS AND FUTURE PERSPECTIVE

158

159

CONCLUSIONS

Procedures for preparing cell walls from L. minor and for extracting the 22°C chelator-
soluble pectic polysaccharides of the cell walls were improved. Thirty-two percent (32%)
of the total pectic polysaccharides (measured as uronic acid) in purified cell walls of L. minor
was solubilized with 0.05 M ammonium oxalate (pH 5.5) at 22°C in 30 min.

A fast, simple yet effective procedure of pectic polysaccharide purification was
developed. Pectic polysaccharides solubilized by 0.05 M ammonium oxalate were directly
applied to a DEAE column whose column buffer was the same as the extraction buffer. All
steps that involved dialysis, concentration, and freeze-drying of polysaccharide solutions
were eliminated so that the sample loss and degradation reactions of polysaccharides were
minimized .

Conditions were established for quantitatively eluting pectic polysaccharides from
DEAE-columns. Cations were found to greatly affect the elution of pectic polysaccharides
from anion exchange columns. Six plant pectic polysaccharides from four different plant
sources (citrus, apple, duckweed, and celery) were quantitatively (or nearly so) eluted from
DEAE-Trisacryl columns by 0.5 M NH4C1 in 0.05 M ammonium acetate buffer (pH 5.5).
In contrast, the elution of five of these six pectic polysaccharides was incomplete when up
to 1 M NaCl or KCl in acetate buffer were used to develop the DEAE columns. N a” and K”
were found responsible for precipitation and gel formation with the tested pectic
polysaccharide in test tubes and apparently also in the DEAE columns. The discovery that

NH], Li+ and Cs” in the eluent resulted in the essentially quantitative elution of pectic

160

polysaccharides from anion exchange resins solves the long-standing problem of incomplete
recovery of pectic polysaccharides from DEAE column chromatography.

Homogeneity with respect to sugar composition, molecular size and degree of
methylesterification was established for the pectic polysaccharide material in column
fractions after the chelator-soluble pectic polysaccharides of L. minor were fractionated by
DEAE chromatography. Analysis of individual DEAE-column fractions by HPSEC-MALLS
resulted in detection of four pectic polysaccharides, PS-I, PS-II, PS-III and PS-IV, with peak
MW ranges of 50,200 - 75,400, 18,800 - 99,700, 24,200 - 150,000 and 6,170 to 2 163,000,
respectively. PS-IIb and PS-IVb, the major portion of pectic polysaccharides PS-II and PS-
IV, respectively, are homogeneous in sugar composition and methylester content, and they
both show a single, virtually symmetrical peak when they were rechromatographed on
DEAE-Trisacryl Plus M columns.

Primary structures were further established for the two purified L. minor pectic
polysaccharides, PS-Ilb and PS-IVb. PS-IIb is a apiogalacturonan with a peak 1171w range of
28,100 - 99,700, and it consists of a a-l,4-1inked D-GalAp backbone with 1,3'-linked
apiobiose as the predominant side chains connected to the 0—2 position of nearly every other
galacturonosyl residues of the backbone. The carboxyl groups of GalA residues are free of
methylester. Besides GalA (54.5%13.2%) and Api (39.3%:t1.4%), small amounts of Rha
(2.3%:0.3%), Xyl (2.5%:tl.4), Ara (1.4%:0.8%) and other common neutral sugars (<0.5%)
were detected by sugar composition analysis of PS-IIb but the sugars present in small
amounts could not be conclusively established as components of PS-lIb. Further degradation

of PS-IIb by poly-[1,4-a-D-galacturonide] glycanohydrolase (EC 3.2.1.15) after side chains

161

containing apiose were removed by mild acid hydrolysis indicated that the sugar detected in
small amount, if not contaminants, are present in the side chains. PS-IIb is somewhat
different than the three types of plant pectic polysaccharides isolated so far
—homogalacturonan, Rhamnogalacturonan-I and Rhamnogalacturonan-H — since apiose
is the predominate neutral sugar residue and the small amount of rhamnose (assuming it is
a component of PS-IIb) was found to be solely terminal-linked.

PS-IVb is basically an a-l,4-linked homogalacturonan and contains 96.3% GalA and
has a peak MW range of 53,300 to at least 163,000. The carboxyl group of the GalA residues
are not methylesterified. Although Api (2.1%), Xyl (1.6%) and trace amounts (<0.5%) of
other neutral sugars such as t-Rha were detected in sugar composition analysis or in
methylation analysis, they appeared to be contaminants rather than components of PS-IVb,
since most (~99%) of PS-IVb was degraded by poly-[1,4-a-D-galacturonide]
glycanohydrolase to GalA without first a mild acid hydrolysis. The isolation of a pectic
polysaccharide of high GalA content basically free of methylester while using mild
conditions has not been reported before. In the presence of N a“ and K“, the polysaccharide
formed what appeared to be a gel and was held on DEAE columns when these ions were in
the eluent. Consequently this type of polysaccharide is probably present in other plants but
has escaped detection because it was not eluted from ion-exchange columns.

A micro-scale method was developed to depolymerize and reduce pectic polysaccharides
before they were converted to alditol acetates for GC analysis. All reactions, including
methanolysis/methyl esterification, carboxyl-reduction, hydrolysis and reduction-acetylation,

. were conducted in an single vial. Compared with other methods tested (1,2), the proposed

162

method gave better depolymerization of the pectic polysaccharides, increased conversion of
uronic acid to neutral sugars, and less sample loss. In addition multiple samples can be

processed simultaneously on a micro-scale (0.1 1-0. 14 mg).

FUTURE PERSPECTIVE

Data collected in this research described the basic structure of PS-IIb (an
apiogalacturonan) and PS-IVb (a homogalacturonan). However, small amounts of neutral
sugars such as Rha, Xyl, and Ara were detected in the polysaccharides by sugar composition
analyses. Also, t-Rhap, t-pentose and 1,4-linked hexose were found in both PS-IIb and PS-
IVb. Are these residues components or contaminants? Most commonly, Rha is found to be
2-linked and 2,4-linked in the backbone of pectic polysaccharides of plants. If t-Rhap is a
component of PS-IIb and PS -IVb, these two polysaccharides are exceptions to this generality.
Is t-Rha linked to Api as a disaccharide side chain? These questions can be answered by
separating and analyzing oligosaccharides fragments containing those sugars from controlled
acid hydrolysis of PS-Ilb and PS-IVb or by selectively degrading the GalA residues of these
two polysaccharides for example by lithium in ethylenediamine (3,4).

Although both PS-lIb and PS-IVb were solubilized from cell walls of L. minor by a
chelator, they are quite different. PS-IVb is a smooth long-chain polymer that lacks branches
and methylester groups while PS-IIb is a highly ramified polymer with mainly apiobiose side
chains evenly distributed along the PGUA backbone. Why do cell walls of L. minor need

both? What is their function? The pectic polysaccharide RG-I has been isolated from many

163

plants and found to have “smooth” and “hairy regions” (5,6). Junction zones are formed in
smooth regions through Ca2+ bridges (7,8) but are spaced by esterification of the carboxyl
groups of GalA residues and by neutral sugar side chains attached to GalA residues so pores
are formed in the cell wall (9). Is the function of PS-lVb similar to that of smooth regions
and the function of PS-lIb similar to that of hairy and esterified regions? Results with some
plants suggests that more junction zones are formed after cell wall elongation (10).
Examining the ratio of PS-IIb and PS-IVb in cell walls of L. minor at different stages of
growth may provide information about the above question. Polyclonal antibodies (anti- RG-I
and anti- xyloglucan) have been used with the electron microscope to locate polysaccharides
in cell walls of plant (1 l). The future research should include trying to raise monoclonal
antibodies against PS-IIb and PS-IVb and determine their location in cell walls of L minor
at different growth stages. Such studies could help us learn when and where these
polysaccharides are synthesized and how they are finally positioned.

Fifty-seven percent (57%) of the total pectic polysaccharides of the cell wall were not
solubilized by the extractant (0.05 M ammonium oxalate) at 22°C. Efforts should be made
to extract and analyze these polysaccharides in order to have a comprehensive knowledge of
the pectic polysaccharides of the cell wall of L. minor.

The procedures successfully developed in this research with L. minor enable us to purify
cell walls, extract and purify pectic polysaccharides with a simplified process, and establish
homogeneity of polysaccharide samples in column fraction of ion-exchange chromatography.
But they all can be used with cell walls from other plants. With the conditions established

in this research, complete elution of the pectic polysaccharides that consist of high GalA

164

units from anion-exchange columns becomes possible. It is desirable to test these procedures
and conditions with cell walls from other plants in order to find if chelator-soluble

homogalacturonans exist widely in higher plants.

10.

11.

165

REFERENCES

Taylor, R. L., and Conrad, H. E. (1972) Biochemistry, 11,1383-1388.

De Ruiter, G. A., Schols, H. A., Voragen, A. G. J. and Rombouts, F. M. (1992)
Anal. Biochem. 207, 176-185.

Mort, A. J. and Bauer, W. D. (1981) J. Biol. Chem, Vol. 257, No. 4, 1870-1875.
Lau, J. M., McNeil, M., Darvill, A., and Albersheim, P. (1987) Carbohydr. Res., 168,
219-243.

Schols, H. A. and Voragen A. G. J. (1994) Carbohydr. Res., 256, 83-59.

Thibaul, J .-F., Renard, C. M. G. C., Axelos, A. V., Roger, P. and Crépeau, M.-J. (1993)
Carbohydr. Res., 238, 271-286.

Morris, E. R., Powell, D. A., Gidley, M. J. and Rees, D. A. (1982) J. Mol. Biol., 155,
507-516.

Rees, D. A. (1975) MTP Int. Rev. Sci. Biochem. Ser. One, Vol. 5, 1-42.

Baron-Eple, 0., Gharyl, P. K. and Schindler, M. (1988) Planta, 175, 389—395.
Carpita, N. C. and Gibeaut, D. M. (1993) The Plant Journal, 3, 1-30.

Moor, P. J ., Darvill, A. G., Albersheim, P. and Staehelin, L. A. (1986) Plant Physiol.,

82, 787-794.

APPENDICES

APPENDIX A

SOLUBILIZATION OF PECTIC POLYSACCHARIDES FROM THE CELL

WALLS OF Lemna minor AND Apium Graveolens

(Reprinted from Phytochemistry)

166

167

0031—9422(95)00675—3 Phylum", Vot st. No. 3. pp. 119—m. ms
Copyright 0 I996 EIsevs'er Sa'enos LII

Printed in Great Britain All rights reserved
(Km-9422M mm + 0.00

@ Pergamon

SOLUBILIZATION OF PECTIC POLYSACCHARIDES FROM THE CELL
WALLS OF LEMNA MINOR AND APIUM GRA VEOLENS

PAUL K. KINDEL. LIANG CHENG and BRAND! R. ADE

Department of Biochemistry, Michigan State University, East Lansing, MI 48824-1319, U.S.A.
(Received in revised form 15 July 1995)

Key Word Index—Lemna minor, Lemnaceae; duckweed; Apium graveolens, Umbelliferae; celery, cell
wall; pectic polysaccharides.

 

Abstract—The kinetics of solubilization of pectic polysaccharides from purified cell walls of Lemma minor (duckweed)
and Apium graveolens (celery) by ammonium oxalate at 22° was determined. With cell walls of L. minor, 30 :1; 1
(average of three experiments, average deviation from the mean) and 34 j; 1% of the total anhydrouronic acid of the
cell walls was solubilized in 15 min and 5 hr, respectively. Water at 22° solubilized 1.3 d: 0.2% and 1.3 1 0.4% of the
total anhydrouronic acid in 15 min and 5 hr, respectively. With cell walls of A. graveolens, 19 j; 1% (average of two
experiments, average deviation from the mean) and 23 i 1% of the total anhydrouronic acid of the cellwall was
solubilized in 15 min and 3 hr, respectively. Water at 22° solubilized 0.9 1 0.6 and 0.9 1; 0.1% of the total anhyd-
rouronic acid in 15 min and 3 hr, respectively. When trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid was
the extractant, recovery values based on weight for the soluble pectic polysaccharide fractions were incorrect (high)
Color formation in the uronic acid test was inhibited by sodium dodecyl sulfate A rapid, two-step procedure for

isolating purified cell walls was developed.

 

INTRODUCTION

The primary cell wall of plants maintains the structure of
growing plant cells and is involved in a variety of impor-
tant biological processes [1]. Pectic polysaccharides‘ are
one of the three polysaccharide constituents of primary
cell walls. Little is known about the structural role of
pectic polysaccharides in cell walls or about their bio-
synthesis. Understanding these requires that the struc-
ture of the pectic polysaccharides be known. The com-
plete structure of any pectic polysaccharide has not been
determined and even less is known about the degree of
structural variation in the total complement of pectic
polysaccharides present in any one plant. In order to
obtain accurate structural information a suitable isola-
tion prowdure must be used.

The amount of pectic polysaccharides solubilized from
cell walls is dependent both on the origin of the cell walls
and on the method of extraction [2-15]. A significant
portion of the cell wall pectic polysaccharides is readily
solubilized by chelating agents and the extent of their
solubilization is easily followed colorimetrically. Surpris-
ingly, the time course of solubilization of pectic polysac-

 

‘Peetic polysaccharidesaredefinedas those containingmain-
1y galacturonic acid units in the main chain (backbone) of the
polysaccharide. 1

charides from purified cell walls at 22° has not been
determined. Experiments of this type will show how
distinct the difference in solubility is between cell wall
pectic polysaccharides readily solubilized and those that
tend to remain in the cell wall. They will also permit
rational selection of the shortest possible extraction
time needed for complete solubilization of a fraction.
Our main goal was to determine the kinetics of solubil-
ization of pectic polysaccharides from purified cell walls
of Lemna minor (duckweed) and Apium graveolens
(celery)

Pure cell walls are needed before the kinetics of solubil-
ization of pectic polysaccharides can be determined. Both
organic [16. 17] and aqueous solvents have been used in
the isolation of cell walls, and both have their disadvan-
tages. When aqueous solvents are used cell wall constitu-
ents may be solubilized or enzymatically degraded or
both. The solubilization of cell wall pectic polysacchar-
ides during the preparation of cell walls has been re-
ported; however, in no case was the solubilized material
firmly identified [2—8]. Ionic detergents such as sodium
dodecyl sulfate (SDS) have been used in the isolation of
cell walls [18], but we found that SDS binds to isolated
cell walls and subsequently interferes with color develop-
ment in the uronic acid assay (this paper). Our second
goal was to develop an aqueous cell wall isolation pro-
cedure that was rapid, mild, simple and yielded pure cell
walls in good yield.

719

168

720

RESULTS

Characterization of cell walls

Cell walls prepared from L minor were dark green
after treatment with a Waring blender but pale green to
white after treatment with both a Waring blender and
a French press. This indicated numerous intact cells with
intact chloroplasts were present after the homogeniza-
tion step but few were present after both steps. Examina-
tion of both preparations by both regular and confocal
light microscopy confirmed that this was the case. Exam-
ination of the final cell wall preparation by electron
microscopy showed only cell walls and an occasional
thick-walled cell were present. Membranes were not ob-
served adhering to cell walls. The final celery cell wall
preparations were cream-colored. Some small multicellu-
lar pieces of tissue were observed in the celery prepara-
tions and based on examination by light microscopy we
estimated 80-85% of the cells were broken. The protein
content of purified cell walls of L minor and A. graveolens
was 5.5% and 1.0%, by weight, respectively. Purified cell
walls from other plants have similar protein contents
[19]. The uronic acid content of the cell walls of L. minor
and A. graveolens was 189 i 10 mg of anhydrouronic
acid per gram of dry cell walls (mean of three experi-
ments) and 251 j; 10 mg per gram (two experiments),
respectively.

Solubilization of cell wall pectic polysaccharides

The average results of three experiments measuring the
time course of solubilization of pectic polysaccharides
from purified cell walls of L minor at 22° with 0.05 M
ammonium oxalate (pH 5.5) are shown in Fig. 1. On
average 30 j: 1% (average deviation from the mean) of
the total anhydrouronic acid of the cell walls was
solubilized in 15 min and 33 j; 1% (two experiments for
this value), 30 i 3%, and 34 i 1% was solubilized in
1,3 and 5 hr, respectively. Water at 22° solubilized
1.3 i 0.2% of the total anhydrouronic acid of the cell
walls in 15 min and 1.3 1; 0.4% in 5 hr. The total amount
of anhydrouronic acid recovered in the 15 min and 5 hr
ammonium oxalate soluble fractions and the correspond-
ing insoluble residue fractions was 99 i 5% and
101 i 4%, respectively. The total cell wall material
solubilized by ammonium oxalate in 15 min and 5 hr was
7.9 i 1.6% and 6.2 i 1.2%, respectively. These values
are based on the dry weights of the starting cell walls and
recovered ammonium oxalate residues.

The average results from two experiments measuring
the time course of solubilization of pectic polysaccharides
from purified cell walls of A. graveolens by 0.05 M
ammonium oxalate (pH 5.5) at 22° showed that
19 11%.22 i 1%,22 11%, and 23 i 1% of the total
anhydrouronic acid of the cell walls was solubilized in
15 min, 1 hr, 2 hr and 3 hr, respectively (Fig. 1). Less than
1% of the total anhydrouronic acid of the cell walls was
solubilized by 15 min and 3 hr extractions with water at
22°. 1n the above experiments, anhydrouronic acid values

P. K. KINDEL et al.

 

 

 

 

 

 

 

:2

s T ‘ 4

g 30‘ i

26

3

'8 1' z —1-

( 20-

3

<

i

3 10-
0‘ r v r r f r r v f—x‘
0 t 2 3 4 5

Hours

Fig 1. Time course of solubilization of pectic polysaccharides
from purified cell walls 1.. minor (average of three experiments)
and A. graveolens (two experiments). Purified cell walls of L.
minor and A. graveolens were extracted with ammonium oxalate
(O. L. minor; A, A. graveolens) and water (0, L. minor; A, A.
graveolens) as described in the Experimental section. The anhyd-
rouronicacidcontcntsofthedrycellwallsandtbemethodof
calculating percent values are given in the Results. Dry weights
of cell walls were calculated from wet weights and the wet
weight/dry weight ratio of the cell walls For L. minor the
average wet-weight/dry-weight ratio of the cell walls was
15.3 :1; 2.4; for A. graveolens it was 12.7 :1: 0.4. Error bars show
the average deviation from the mean. AUA, anhydrouronic acid.

were normalized on the basis of 1 g dry weight of cell
walls extracted and then percentage values were cal-
culated. Percent values are relative to the anhydrouronic
acid content of dry cell walls, which was set equal to
100%.

Analysis of filtrates from the preparation of cell walls for
uronic acid

The amount of anhydrouronic acid in the combined.
dialysed filtrates obtained during the preparation of cell
walls of L. minor and A. graveolens was 13% (three
experiments) and 5.0% (two experiments), respectively. of
the total anhydrouronic acid present in the combined
filtrates plus cell walls. Percent values were calculated
from anhydrouronic acid values normalized on the basis
of 1g dry weight of cell walls extracted.

Efl’ect of various chemicals on the uronic acid test

HCl (1 M), NaCl (1 M) and ammonium oxalate
(0.05 M) present individually in samples containing
known amounts of galacturonic acid had no effect on
color formation in the uronic acid test; control sample!
were in water. In contrast, 0.0031% and 0.031% (w/V)
SDS present in a galacturonic acid solution inhibited
color formation 39 and 94%, respectively.

When SDS was used in the isolation of L. minor cell
walls, measurements of uronic acid in the ammonium
oxalate extract were low. Whole plants were homogen-
ized and to one half of the homogenate SDS was added
to a concentration of 0.52% (w/v) and to the other half
water was added. After stirring for 15 min and centrifus'

169

Solubilization of pectic polysaccharides

ing, the cell wall fraction was washed once with 0.1 M
NaCl and once with water and then extracted twice with
ammonium oxalate. each time for 4 hr. The amount of
uronic acid detected in the ammonium oxalate soluble
extracts from cell walls prepared with SDS present, per
gram dry weight of cell walls, was 44% (average of two
experiments) of that in the water control.

Extraction of cell walls with trans-1,2-diaminocyc-
lohexane-NWN'N'detraacetic acid (CDTA)

Individual samples of cell walls of L. minor were ex-
tracted with CDTA and ammonium oxalate for 15 min
and 5 hr at 22°. The chelator-soluble fractions were dialy-
zed for 21 hr in water with six changes and dried to
constant weight. The dry weight of the 15 min and 5 hr
fractions solubilized by CDTA was 4.7 times and 4.8
times greater, respectively, than the corresponding am-
monium oxalate fractions. The values were normalized
on a per gram dry weight ofcell walls extracted. The
anhydrouronic acid and total sugar content of the two
extracts obtained at each extraction time were basically
the same.

DWON

The results in Fig. 1 show there is a rapid solubiliza-
tion ofpectic polysaccharides from purified cell walls of
L. minor and A. graveolens by ammonium oxalate at 22°
and then the rate of solubilization decreases sharply until
solubilization stops. The time needed for complete
solubilization of the 22° ammonium oxalate-soluble frac-
tion was approximately 30 min; there was, however, only
a slight increase in the percent solubilized between
15 min and 1 hr (Fig. 1). The almost complete cessation
of solubilization showed that the difference in solubility
between the soluble and insoluble pectic polysaccharides
is distinct. When impure preparations of cell walls of L.
nrinor were used (preparations obtained following the
Waring blender step). the rate of solubilization was not as
rapid as that shown in Fig l and pectic polysaccharides
continued to be released for the entire 5 hr (data not
presented) This result shows the importance of using
purified cell walls. We conclude that the chelator-soluble
pectic polysaccharides are held in the cell wall by ionic
interactions alone.

Extraction times of 125—6.5 hr have been used to
solubilize the 22° chelator-soluble cell wall fraction [2, 8,
9, 12. 14]. Our results show that such long extraction
times are unnecessary and are probably undesirable be-
cause of the possibility of polysaccharide degradation.
The results in Fig. 1 show that a 30 min extraction with
ammonium oxalate at 22° is sufficient to solubilize the
chelator-soluble fraction, at least with the two cell walls
tested. Since a rapidly-solubilized pectic polysaccharide
fractionispresentinbothamonocotandadicouthis
typeoffractionmaybepresentinthecellwsllsofmany
higher plants.

The extent to .whieh cell wall pectic polysaccharides
aresolubilizedduringthepreparationofcellwallsisnot

721

known. Our results with L. minor and A. graveolens show
that 13% and 5.0%, respectively, of the total uronic acid
units present in the cell walls plus filtrates were present in
the filtrates. It was not established if the uronic acid
material in the filtrates originated from the cell walls.
However, no significant amounts of pectic polysacchar-
ides were solubilized by water once the cell walls were
isolated (Fig. l)

HCl and NaCl at 1 M and ammonium oxalate at
0.05 M in the sample solution do not interfere, with color
formation in the uronic acid test, but SDS does. CDTA,
when used as the extractant, was not completely removed
from the 22° chelator-soluble fraction by dialysis in water
and therefore incorrect (high) dry weights of the fraction
were obtained (data not presented). Similar results have
been reported by others [20].

The findings that the 22° chelator-soluble pectic poly-
saccharide fraction of plant cell walls was solubilized in
a short time - slightly more than 15 min - and that
solubilization of cell wall material virtually ceased after
this time period have not been reported previously. The
time needed for solubilization of the 22° chelator-soluble
fraction was the same for the two plants. In each plant
a specific percentage of the total cell wall pectic polysac-
charides was solubilized, however, the percent solubilized
was different for the two plants. These findings were
obtainedonlywhenpurifiedcellwallswereused.

EXPERIMENTAL

Materials and general methods. Lemna minor was
grown as dacribed elsewhere [21]. Apium graveolens was
purchased from a local grocery. Dialysis tubing with
a 6000—8000 M, cutoff was from Spectrum Medical In-
dustries, Inc. Nylon mesh (3—15/6) was from Tetko, Inc.
Uronic acid and total sugar were determined with 3-
hydroxydiphenyl [22] and phenol-sulfuric acid [23], re-
spectively. The determinations of uronic acid and neutral
sugar in the combined filtrates were corrected for mutual
interference. Equations based on Beer’s law were derived
for calculating the concn of each sugar type measured in
the presence of the interfering sugar type. For the calcu-
lations. D-galacturonic acid monohydrate and sucrose
were used as the representative uronic acid and neutral
sugar types, respectively. The extinction coefficients of
these two in both sugar tests were determined and were
used in the equations. Bovine serum albumin was used as
the protein standard Nitrogen by the Kjeldahl method
was determined by Galbraith Laboratories and the pro-
tein content was calculated by multiplying by 6.25. Sam-
ples were dried to constant weight under vacuum and
over P30, at 22°. Cell wall prepns were examined by
transmitted brightfield microscopy with a standard light
microscope and those from L. minor were further exam-
ined by laser scanning transmitted brightfield micros-
copy with a Zeiss 10 laser scanning microscope. Cell wall
prepns of L. minor were also examined by transmission
electron microscopy at 10000 to 59 000 x with a Phillips
CM to electron microscope operated at 100 kV. Samples
were fixed with glutaraldehyde and 0.04 and infiltrated

170

1n

with Quetol 651-vinylcyclohexane dioxide [24]. The
sample sections were 90 nm thick and were stained with
uranium acetate and lead citrate.

Preparation of cell walls and rate of solubilization of
pectic polysaccharides. An experiment with L minor is
described; however, the same procedure was used with A.
graveolens except one extraction time was different.
Whole plants of L minor were suspended in water at 22°
and the water was decanted Washing was repeated four
times. Plants were freed of excess water with absorbent
paper and weighted wet (52 g). Petioles of A. graveolens
were diced at 4° to cubes 0.5—1.0 cm on a side.

The following was performed at 4°. Plants (47.7 g, wet
wt) were suspended in 120ml of 1.0 M NaCl and hom-
ogenized for four, 40-s periods in a Waring blender with
10sec intervals between periods. The suspension was
filtered with 15 um Nylon mesh, the particulate material
was washed with 75 ml of water and the combined fil-
trates were saved. The cell walls were resuspended in
120ml of water and passed through a French pressure
cell at an average cell pressure of 16000 pounds/in’. The
suspension was filtered through 15 um Nylon mesh, the
cell walls were washed with 75 ml of water and the
combined filtrates were saved. The cell walls were
weighed (9.3805 g, wet wt) and examined microscop-
ically.

Two weighed portions of cell walls, each 0.4 1; 0.04 g,
wet wt, were dried to constant weight and two weighed
portions, each 0.3 1; 0.04 g, wet wt, were hydrolysed as
described below. The remainder of the cell walls was
divided approximately equally between six, 50 ml
beakers (each sample was about 0.85 :1; 0.05 g, wet wt)
and the wet weights recorded. Four of the cell wall
portions were suspended in 0.05 M ammonium oxalate
(pH 5.5) (8.5 ml per sample) and stirred for 15 min, I, 3,
and 5 hr at 22°. Two portions were suspended in water
(8.5 ml per sample) and stirred for 15 min or 5 hr at 22°.
The suspensions were centrifuged at 4° and the super-
natant solutions were filtered The residues were sus-
pended in water and the suspensions were centrifuged
and the supernatant solutions filtered The washing was
repeated once and the three filtrates were combined and
analysed for uronic acid

One weighed portion of the washed residue from both
the 15 min and 5 hr ammonium oxalate extractions, each
approximately 0.3 g, wet wt, was hydrolysed and a sec-
ond weighed portion (about 0.4 g, wet wt) of each was
dried to constant weight. Hydrolysis of samples was by
refluxing with 20 ml of 0.25 N HCl for 3 hr at 100° [25].
The hydrolysed samples at 22° were brought to pH
7 with 2.0 N KGB/0.5 M ammonium oxalate and stirred
for 10 min. The samples were centrifuged at 4° and the
supernatant solutions were filtered. The precipitates
were resuspended in water, the suspensions filtered, and
the appropriate filtrates combined and analyzed for
uronicacidTherecultswerenormalizedonapergram
drywtofcellwallsbasisandthenconvertedtopercent
(Pic 1)

Analysis of the combined filtrates from the preparation
of cell walls for sugars. The volumes of the two combined

P. K. Kmnar. er al.

filtrates obtained in the above procedure for the prepn of
cell walls were measured. A portion of each was centri-
fuged and the supernatant solutions were filtered
through separate 5.0 pm MF-Millipore filters. The two
samples were dialysed in water for 26 hr, their volumes
were measured and they were assayed for uronic acid and
total sugar.

Acknowledgements— We thank Drs Joanne Whallon and
John W. Heckman of the MSU Laser Scanning Micro-
scope Laboratory and the Center for Electron Optics,
respectively, for microscope work.

RUBWGS

1. Carpita, N. C., and Gibeaut, D. M. (1993) Plant 3, l.

2. Jarvis, M. C. (1982) Planta 154, 344.

3. Stevens. 3. J. H. and Selvendran, R. R. (1980) J. Sci.
Food Agric. 31, 1257.

4. Ring, 8. G. and Selvendran, R. R. (1981) Phytochem.
N, 2511.

5. Stevens, B. J. H. and Selvendran, R. R. (1984) Carbo-
hydr. Res. 128, 321.

6. Stevens, 8. J. H. and Selvendran, R. R. (1984) Carbo-
hydr. Res. 135, 155.

7. Redgwell, R. J. and Selvendran, R. R. (1986) Carbo-
hydr. Res. 157, 183.

8. Ryden, P. and Selvendran, R. R. (1990) Biochem. J.
269, 393.

9. Souty, M., Thibault, J.-F., Navarro-Garcia, G.,
Lopez-Roca, J.-M. and Breuils, L (1981) Sci. des
Alim. 1, 67.

10. De Vries, J. A., Voragen, A. G. J., Rambouts, F. M.
and Pilnik, W. (1981) Carbohydr. Polymers l,
117.

11. Aspinall, G. O. and Fanous, H. K. (1984) Carbohydr.
Polymers 4, 193.

12. Sauliner, L and Thibault, J.-F. (1987) Carbohydr.
Polymers 7, 329.

13. Marcelin, 0., Sauliner, L and Brillouet, J.-M. (1991)
Carbohydr. Res. 212, 159.

14. Ryden, P. and Selvendran, R. R. (1990) Carbohydr.
Res. 195, 257.

15. Jarvis, M. C., Hall, M. A, Threlfall, D. R. and Friend,
J. (1981) Planta 152, 93.

16. Selvendran, R. R. (1975) Phytochem. I4, 1011.

17. Selvendran, R. R. and DuPont, M. S. (1980) J. Sci.
Food Agric. 31, 1173.

18. Selvendran, R. R. and O'Neill, M. A. (1987) in
M ethods of Biochemical Analysis (Glick, D., ed), Vol.
32, p. 25, John Wiley and Sons, New York.

19. Harris, P. J. (1983) in Isolation of Membranes and
Organelles from Plant Cells (Hall, J. L. and Moor?»
A. 1..., eds), p. 25, Academic Press, New York.

20. Mort, A. J., Moerschbacher, B. M., Pierce. M. L. and
Maness, N. O. (1991) Carbohydr. Res. 215, 217.

21. Kindel, P. K. and Watson, R. R. (1973) Biochem. J.
133, 227.

22.

23.

171

Solubilization of pectic polysaccharides 723
Blumenkrantz, N. and Asboe-Hansen, G. (1973) 24. Kushida, H. (1980) J. Electron Microsc. 29.
Anal. Biochem. 54, 484. 193.

DuBois, M., Gilles, K. A., Hamilton, J. K., Rebers, 25. Jarvis, M. C., Forsyth, W. and Duncan, H. J. (1988)
P. A. and Smith, F. (1956) Anal. Chem. 28, 350. Plant Physiol. 88, 309.

APPENDIX B

REEXAMINATION OF THE ACETYLATION OF APIITOL

IN THE DETERMINATION OF APIOSE

(Reprinted from Carbohydrate Research)

172

173

 

 

CARBOHYDRATE
RESEARCH

 

 

Carbohydrate Research 290 ( 1996) 67—70

 

Note

Reexamination of the acetylation of apiitol in the
determination of apiose

Liang Cheng, Paul K. Kindel '

Department of Biochemistry. Michigan State University. East Lansing, MI 48824-1319. L'SA

Received 22 January 1996; accepted in revised form 27 April 1996

 

Keywords: Apiose; Apiitol; Xylose; GLC

 

Apiitol, the sugar alcohol of apiose [3-C-(hydroxymethyl)-D-g1ycero-aldotetrose]. is
considerably more difficult to acetylate completely than sugar alcohols with only
primary and secondary hydroxyl groups. We attempted to acetylate apiitol completely by
modifying the conditions used by Blakeney et al. [1] for other sugars. When acetylation
was performed at 35 °C for 1—20 h, a side-product, 1,2.4-tri-0-acetyl-3-C—(acetoxy-
methyl)-3-0-(methylthiomethyl)-D-glycero-tetritol [herein called 3-0-
(methylthiomethyl)apiitol tetraacetate], was formed in substantial amounts [2]. Replacing
dimethylsulfoxide (Me2SO) with dimethylforrnamide avoided the formation of the
side-product [2]. Harris et al. [3] reported that the methylthiomethyl ether side-product
was not observed when the procedure of Blakeney et al. [1] was used with Mezso and
the acetylation of apiitol was conducted at 40 and 80 °C for 90 min. In an attempt to
reconcile the difference in results we used the conditions of Harris et al. [3] to acetylate
apiitol. The methylthiomethyl ether side-product was again observed. At 40 and 80 °C
the ether side-product was 30 and 38%, respectively, of the total peak area of the three
compounds derived from apiose. The results at 40 °C were similar to those obtained
previously at 35 °C [2]. Small amounts of the methylthiomethyl ether (0.2—1.8%) were
detected even when acetylation was performed at 22 °C for 10 min. The amounts were
dependent on how quickly the sample was cooled back to 22 °C after acetic anhydride
was added. These results were obtained with the apiose used in the previous investiga-
tion [2] and with apiose purchased as the diisopropylidene derivative. Subjecting apiose
to hydrolysis conditions [2 M trifluoroacetic acid (TFA). 120 °C, 1 h] before acetylation

‘ Corresponding author.

0008-6215/96/31500 Copyright © 1996 Elsevier Science Ltd. All rights reserved.
PII 80008-6215(96)00128-0

174

 

 

 

 

 

 

 

 

 

 

 

68 L Cheng. P. K. Kindel / Carbohydrate Research 290 (I996) 67- 70
2 3
0)
(0 l 1 4
8 . 11
3
a) 7 8
CI 10
L-
o F
o—s
O
Q)
o—s
d)
C) 9
1M . UL

 

0 2 4 6 8 10 12 14

Time (min)
Fig. l. Gas—liquid chromatogram of alditol peracetates and related compounds. The sample was prepared by
the procedure of Blakeney et al. [1] except that the acetylation was performed at 40 °C for 90 min, and the
extracted sample in dichloromethane was washed with 5 mL of water three times [11,12]. The sample was
analyzed with a DB-225 capillary column operated isothermally at 230 °C. Peaks represent the peracetates of
the following alditols, except as noted: I, rhamnitol; 2, fucitol; 3, arabinitol; 4, xylitol; 5, apiitol; 6, apiitol

tetraacetate; 7, mannitol; 8, galactitol; 9, 3-0-(methylthiomethyl)apiitol tetraacetate; 10, glucitol; 11, myo-in-
ositol hexaacetate.

had no influence on the formation of the three products. The above results are consistent
with our earlier results [2] but are contradictory to those of Harris ct al. [3].

Harris et al. [3] stated that “the acetylation of sulphoxides in the Pummerer reaction
is well documented, but side products are not formed when the reaction is conducted
below 80°.” Results in the literature do not support this as a general statement. The
Pummerer reaction involving MeZSO and acetic anhydride was shown to occur at 25 °C
with formation of the expected sulfide [4]. The formation of the methylthiomethyl ether
of a variety of alcohols, including sugars, from MeZSO and acetic anhydride was also
shown to occur at room temperature [5—9]. The rate of reaction was sufficient to detect
the methylthiomethyl ether by GLC after 10 min. These results support our observation
that the methylthiomethyl ether side-product was formed when more rigorous acetyla-
tion conditions were used in the procedure of Blakeney et al. [1].

Apiose can be determined quantitatively by measuring the peak area of apiitol
pentaacetate after GLC [2]. Separation of apiitol and xylitol pentaacetates by GLC is
difficult and a long sample separation time was required [2,3]. The method has been
improved by reducing the separation time to less than 15 min while maintaining almost
complete separation of apiitol and xylitol pentaacetates as well as complete separation of
the other acetylation products (Fig. 1). For most samples, a 03-225 capillary column
operated isothermally at 230 °C will give satisfactory results (Fig. 1). When a new
DB-225 capillary column was used at 230 °C, separation of apiitol and xylitol pentaac-
etates was virtually complete (less than 1% of the total area of the two peaks

175

L Cheng, P.K. Kindel/ Carbohydrate Research 290 (I996) 67— 70 69

overlapped). The DB-225 column lost efficiency for separating the two pentaacetates
very slowly. After hundreds of hours of use over 3.5 years, approximately 4% of the
total area of the apiitol and xylitol pentaacetate peaks overlapped under the above
chromatography conditions; however, there was substantial tailing of apiitol tetraacetate.
The high column temperature was needed to separate the apiitol and xylitol pentaac-
etates. For samples where complete separation of apiitol and xylitol pentaacetates is
necessary, both the DB-225 column Operated at a lower temperature and a SP-2380
column gave baseline separation of these two pentaacetates as well as the other
acetylation products stated in the legend of Fig. 1. The DB-225 column was operated
isothermally at 170 °C for 55 min and then the temperature was increased at 1.5 °C / min
to 220 °C and held. Under these conditions the order of elution of apiitol and xylitol
pentaacetates was the reverse of that obtained when the column was Operated isother-
mally at 230 °C (Fig. 1). The order of elution of the other products remained as shown
in Fig. l. The column chromatography time needed for a single sample was approxi-
mately 110 min. Baseline separation of the eight alditol peracetates plus myo-inositol
hexaacetate was also achieved with a SP-2380 capillary column at 260 °C in 12 min.
However, the SP-2380 column lost efficiency relatively rapidly (detectable after approx-
imately 100 h of use) when operated at this temperature.

Harris et al. [3] proposed a method for quantitatively determining apiose based on the
peak area of apiitol tetraacetate after GLC. Their method is more involved than the one
based on apiitol pentaacetate and small amounts of the ether side-product may be
formed.

1. Experimental

Sample preparation—Apiose was isolated from parsley apiin [10]. 1,2:3,5-Di-0-iso-
propylidene-a—D-apiose was purchased from Pfanstiehl. MeZSO, acetic anhydride, 1-
methylimidazole, TFA and sodium borohydride from Sigma or Aldrich, and acetic acid
and ammonium hydroxide were purchased new. Apiose (0.76 mg) and l,2:3,5-di-0-iso-
propylidene-a-D-apiose (0.81 mg) were hydrolyzed with 2 M TFA at 120 °C for 1 h and
the acid was removed with N2 at 22 °C. Apiose (0.76 mg). hydrolyzed apiose, and
hydrolyzed 1,2:3,5-di-0-isopropylidene-a-D—apiose were reduced separately and the
alditols were acetylated by the method of Blakeney et a1. [1], except that the acetylation
was conducted at 22, 40, and 80 °C for 10 and 90 min. For samples acetylated at 22 °C,
the vials were quickly cooled back to 22 °C by an air stream after acetic anhydride was
added. A standard sample containing 0.11-0.12 mg of each of rhamnose, fucose,
arabinose, xylose, mannose, galactose, glucose, and myo-inositol and 0.46 mg of
l,2:3,S-di-0-isopropylidene-a-D-apiose was hydrolyzed with 2 M TFA at 120 °C for l h
and the acid was removed as described above. The sugars and myo-inositol in the
hydrolyzed standard were converted to products with the method of Blakeney et al. [1]
except the acetylation was conducted at 40 °C for 90 min, and the extracted sample in
dichloromethane was washed with 5 mL of water three times [11,12].

Gas chromatography. —Compounds were chromatographed on a DB-225 column [30
m x 0.25 mm (i.d.), 0.15 pm film thickness; J.&W.] attached to a Hewlett—Packard gas

176

70 L Cheng. P. K. K indel / Carbohydrate Research 290 ( I996) 67— 70

chromatograph, Model 5840A, equipped with a splitter, a flame-ionization detector, and
a 5840A data terminal. Helium was used as the carrier gas. When chromatography with
the DB-225 column was performed isothermally at 230 °C, the sample size ranged from
1.2 to 4 pl... the carrier gas flow rate was 1.4 mL/ min, and the split ratio was 1:5 (for
apiose samples acetylated for 10 min at 22 °C) or 1:15 (other samples). For chromatog-
raphy starting at 170 °C, the DB-225 column was kept at 170 °C for 55 min and then the
temperature was increased at 1.5 °C / min to 220 °C and held. The sample size was 0.4
11.1., the carrier gas flow rate was 1.7 mL/ min, and the split ratio was 1:15. Compounds
were chromatographed on a SP-2380 column [30 m X 0.25 mm (i.d.), 0.2 pm film
thickness; Supelco] attached to the same system. Chromatography was performed
isothermally at 260 °C. Helium was used as the carrier gas at a flow rate of 1.5
ml. / min. Apiitol tetraacetate, apiitol pentaacetate and 3-0-(methylthiomethyl)apiitol
tetraacetate were identified by their retention times from GLC and by their mass spectra
[2]. Mass spectrometry was performed as described previously [2].

References

[l] A.B. Blakeney, PJ. Harris. R.I. Henry, and B.A. Stone, Carbohydr. Res., 113 (1983) 291—299.
[2] P.K. Kindel and L. Cheng, Carbohydr. Res., 199 (1990) 55—65.
[3] PJ. Harris, M. Sadek, R.T.C. Brownlee, A.B. Blakeney, J. Webster, and B.A. Stone, Carbohydr. Res.,
227 (1992) 365-370.
[4] S. an. T. Kitao, S. Kawamura, and Y. Kitaoka. Tetrahedron, 19 (1963) 817—820.
[5] K. James. AR. Talchcll, and (in pan) P.K. Ray, J. Chem. Soc., C. (1967) 2681-2686.
[6] P.M. Pojer and SJ. Angyal. Aust. J. Chem, 31 (1978) 1031—1040.
[7] J.D. Albright and L. Goldman. J. Am. Chem. Soc., 87 (1965) 4214-4216; 89 (1967) 2416-2423.
[8] K. Antonakis and F. Leclercq, Bull. Soc. Chim. Fr., (1971) 4309—4310.
[9] K. Yamada, K. Kato, H. Nagase, and Y. Hirata, Tetrahedron Lett., (1976) 65-66.
[10] 0.1.. Neal and P.K. Kindel, J. Bacteriol.. 101 (1970) 910-915.
[1 l] C. Hocblcr, J.L. Barry. A. David. and J. Delort-Laval, J. Agric. Food Chem. 37 (1989) 360-367.
[12] L. Cheng, M.S. thesis. Department of Biochemistry, Michigan State University, East Lansing, Michigan.
1991.

APPENDIX C

DERIVATION OF EQUATIONS FOR CALCULATION OF THE AMOUNT OF

NEUTRAL SUGAR AND URONIC ACID IN SAMPLES

177

178

DERIVATION OF EQUATIONS FOR CALCULATION OF THE AMOUNT
OF NEUTRAL SUGARS AND URONIC ACID IN SAMPLES

I. BASIC EQUATION

If the concentraion of a substance in solution has a linear relationship with absorption,
the linear equation for the standard curve is:

A = b + me or c = (A-b)/m (1)
where A = absorbance

b = intercept of the standard curve

m = slope of the standard curve

c = the concentration of the substance

II. CONCENTRATION OF NEUTRAL SUGAR IN A SAMPLE MIXTURE WITH
NEUTRAL SUGAR AND GALACTURONIC ACID

For a sample mainly conatining GalA, Ga] and Api, the concentration of Api can be
calculated from absorbance in total sugar test:

cApi :(cGalA +531)“ =MLD1
m1 (2)
where CAP, = the concentration of Api (mg/mL)
cad“ = the concentration of GalA (mg/mL)
c" = the concentration of neutral sugars (Api and Gal)
x = percentage (w/w) of Api in the sample
A490”,- = absorbance of Api in sample in the total sugar test
ml and bl = SIOpe and intercept of the standard curve of Api concentration vs.
absorbance for the total sugar test
D1 = fold of dilution of the sample for the total sugar test

Rearrange (2), the absorbance of Api in the total sugar test is:

(00am +c )ur'm1
490Api_ D: +b1 (3)

A

 

- The concentration of Gal can be calculated by:

179

A49OGal-b2
CGalzcn —CApi =0" ‘(CGaM +Cn)'x :TDI (4)
where cc“, = the concentration of Gal (mg/mL)
A4906“, = absorbance of Gal in sample in the total sugar test
m2 and b2 = slope and intercept of the standard curve of Gal concentration vs.

absorbance for the total sugar test

Rearrange (4), the absorbance of Ga] in the total sugar test is:

[Cu _(CGalA +Cn)'X] .mZ
A49OGal : D + 2 (5)
I

 

The concentration of GalA can be calculated by:

A49OGalA -b
3
CGalA =——'Di (6)
m3
where A4906“ = absorbance of GalA in sample in the total sugar test
m3 and b3 = slope and intercept of the standard curve of GalA concentration vs.

absorbance for the total sugar test
Rearrange (6), the absorbance of GalA in the total sugar test is:

cGaIA

D 1 (7)

A =b3 +m3-

 

49OGalA

The total absorbance of the sample in total sugar test, A490, , can be expressed by combination
of equation (3), (5) and (7):

A490: :A490Api +A49OGal +A49OGalA

: (cGalA +cn).x.ml +b + [cu-(c0011! +6'n).x].mZ

cGalA
1 +b2 +b3 +”’3"—‘ (8)
DI D1

1

 

 

180

Rearrange (8), the concentration of neutral sugar in the sample is:

(A 4905b] -b2 -b3)-Dl -(m3 +x'ml -x°m2)'cGaM

_x.,,,2) (9)

I!

 

(m2 +Jc-m1

III. CONCENTRATION OF GALACTURONIOC ACID IN A SAMPLE MIXTURE
WITH NEUTRAL SUGAR AND GALACTURONIC ACID

Similar to equation (2), in a sample mainly conatining GalA, Gal and Api, the concentration
of Api can also be calculated from absorbance in uronic acid test:

A .-b
_ . _ 52w: 4.
CAP: 4000“ +0..) x-T D2 (10)
where A520”,- = absorbance of Api in sample in the uronic acid test
m4 and b4 = slope and intercept of the standard curve of Api concentration vs.
absorbance for the uronic acid test
D2 = fold of dilution of the sample for the uronic acid test

Rearrange (10), the absorbance of Api in the uronic acid test is:

 

(c +c )°x-m
_ 00“ ll 4+b

A520Api_ D2 4 (11)
The concentration of Ga] can be calculated by:
6001 =cn -CApi :Cn -(CGalA +611)“ LEE—5:51:30 2 (12)
where A520“ = absorbance of Gal in sample in the uronic acid test
m5 and b5 = slope and intercept of the standard curve of Gal concentration vs.

absorbance for the uronic acid test

Rearrange (12), the absorbance of Gal in the uronic acid test is:

181

_ [Cu _(cGalA +99%] ”"5

 

 

A - +b (13)
52000! 5
Dz
The concentration of GalA can be calculated by:
ASZOGalA-b6
Com 2 ——'D 2
m6 (14)
where A5206,“ = absorbance of GalA in sample in the uronic acid test
m6 and b6 = slope and intercept of the standard curve of GalA concentration vs.
absorbance for the uronic acid test
Rearrange (14), the absorbance of GalA in the uronic acid test is:
c
A =b +m ' Ga“
SZOGaZA 6 6
02 (15)

The total absorbance of the sample in uronic acid test, A520, , can be expressed by
combination of equation (11), (13) and (15):

A520: :A520Arn' +A520001 +A520601A

 

 

 

 

= (60‘1“ +cn)'x°m4 +b4 + IC" —(CG"“ +69%] -m5 +b5 +176 +m5- ‘30an (16)
D2 Dz 2
Rearrange (16), the concentration of neutral sugar in the sample is:
C = (Aszox'b4'bs 'b6)°D2_(m6 +x°m4 -x-m5)°cGaM
" (m5 +Jc-m4 -x'm5) ( 17)

Compare equtions (9) and (17), the left side of the equations are equal:

(A490: 'b 1 'b 2 ’b3)'D 1 -(m3 ””1 -x°m2)'cGaM _ (Aszor’b4 "b5 ’b6)'D2 '(me +x°m4 -x°m5)°cGaM

 

(m2 +x°ml -x°m2) (m5 +Jt°m4 -x°m5)

182

(18)

Rearrange equation (18), the concentration of GalA is:

c : (m5 +x-m4 -x-m5)°(A490, ’b 1 'b2 'b3)'D 1 -(m2 +“‘C'ml -x-m2)-(A520, ‘b4 ’b5 “b6)'Dz
0”” (m5 +X'm4 -x-m5)°(m3 +Jc"m1 ~x°m2) -(m2 +Jt-m1 ~x'm2)°(m6 +Jc'm4 ~x'm5)

(19)
Let A = (m5 + x'm4 - x'm5)

C = (m3+ x'm, - x'mz)
D = (m, + x'm4 - x'm5)

then equation (19) is:

(”Amer—b r "bz-b3)°D 1 "B'(A520:-b4 “b5 "b6)°D 2
A-C-B-D (20)

CGalA :

and equation (9) is:

((1490be ”b2 "b3)'D1-C°CGalA
B (21)

n

 

Assume all intercepts = 0, equations (20) is:

= A-A4901-D 1 ’3 ”4520(1) 2
A-C-B-D (22)

 

cGalA

and equation (21) is:

= A4901'D1 ' ocean (23)
n B

 

Equation (22) and (23) were used to calculate GalA and neutral sugars in the sample.

APPENDIX D

A COMPUTER PROGRAM FOR CALCULATION OF THE AMOUNT OF

NEUTRAL SUGAR AND URONIC ACID IN SAMPLES

(By Linag Cheng, 1993)

This program was written with Q-BASIC computer language based on the equations of

APPENDIX C. A IBM compatible PC with a Q-BASIC software is required to run this

program.

183

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APPENDIX E

DERIVATION OF THE EQUATION USED TO CALCULATE THE

GALACTITOL ACETATE-D2 IN GALACTITOL ACETATE

(By Liang Cheng, 1993)

190

191

DERIVATION OF THE EQUATION USED TO CALCULATE THE

GALACTITOL ACETATE-D2 IN GALACTITOL ACETATE

The ratio of GalA/Gal in a pectic polysaccharide is determined from mass spectra of
galactitol acetate-Dygalactitol acetate after the carboxyl groups of GalA residues were reduce
with NaBD4 and the sample was depolymerized and acetylated. Ions with 5 different mass
should be found in the typical isotope cluster of a nominal fragment ion in a mass spectrum
of galactitol acetate-Dzlgalactitol acetate derived as above . The ions in the cluster are shown

in the diagram:

H
N

 

 

 

ll
3
1° JDLFI
: A
> 5
ti :
U) a
Z i
: loo
1, 5
Z l
9 T :
s 1,
ID i
: T
1 $1 F2
D E : i
101:: g ‘L EIDDFI 14
it E : T
L 1on E : IDDFZ

 

 

where IO = the intensity of the nominal mass fragment ion.

192

I1 = the intensity of the fragment ion is one mass unit larger than I0 (the species
with only 'H, 12C, or 18O) which includes the following isotopic species:

10F, = the additional one mass unit due to the presence of one atom of 2H, 13C,
or 17O in the fragment ion.

ID = the additional one mass unit due to one deuterium atom introduced in
to the molecule by N aBD4.

12: the intensity of the fragment ion is two mass unit larger than I0 which includes
following isotopic species:

1on = the additional two mass units due to the presence of two atoms of 13C
or one atom of "‘0 in fragment ion.

IDD = the additional two mass units due to two deuterium atoms introduced in
to the molecule by NaBD4.

IDF1 = the additional two mass units due to the presence of one atom of 2H, 13C,
or 17O in the fragment ion plus one deuterium atom introduced by N aBD4.

I3 = the intensity of the fragment ion is three mass units larger than I0 which
includes following isotopic species :

IDDFl = the additional three mass units due to the presence of one atom of 2H,
13C, or 17O in the fragment ion plus two deuterium atom introduced by
NaBD4.

1sz = the additional three mass units due to the presence of two atoms of 13C
or one atom of '80 in the fragment ion plus one deuterium atom

introduced by NaBD4.

L = the intensity of the fragment ion is four mass units larger than Io which includes
following isotopic species:

IDDF2 = the additional four mass units due to the presence of two atoms of 13C
or one atom of 18O in fragment ion plus two deuterium atoms introduced
by NaBD4.

Since galactitol acetate-D2 has a symmetrical structure (not considering the deuterium

atoms), then for any type of fragment ion with two introduced deuterium atoms there must

193

be a corresponding fragment ion with the same structure except without the introduced
deuterium atoms. Therefore, in above ion cluster, the total intensity of the ions from
galactitol acetate-D2 is: 2 x (IDD + IDDFI + Inon)- The ratio (R) of galactitol acetate-
D2/(galactitol acetate-D2 + galactitol acetate) can be calculated as follows:

R= 2(100+ DDF 1 +1001: 2) = 2°(IDD+IDD°F 1 +100": 2) _ 2100'“ +F1+F2) (l)
(10+Il+12+13+14) (104.11 +12‘LI3J’I4) (10+Il+12+13+l4)

 

where F l is the expected abundance ratio of a fragment ion that is one mass unit lager
due to the natural occurrence of a 2H, 13C, or 170 compared to the same ion without
the natural isotope.

F 2 is the expected abundance ratio of a fragment ion that is two mass unit lager due
to the natural occurrence of two 13C or one ‘80 compared to the same ion without

the natural isotope.

Since I1 = Io'Fl + ID, 12 = IO'F2 + [DD + ID-Fl, I3 = 100 F; + ID-F2 and I4 = IDD'F2 , replace 1,,
12, I3 and I4 in equation (1) by their constituent ion intensities:

2'IDD~(1 +F1+ 2)

 

 

 

R: 2
[10+(10.F1+ID)+(IO.F2+IDD+ID.F1)+ IDD'FI+119°F2)+IDD'F2] ( )
Rearrange (2):
R: 2-(1 ”'1’. +172)
Io-(l -F1-F2)+Il-(l +12) +12 ”'3 +F2 (3)
100

where 100 can not be measured directly but it can be calculated as follows:
Ior) = I2 ‘ Io'Fz ' ID'FI (4)
where [D can not be measured directly but it can be calculated as follows:

ID = I, - 1.,-F, (5)

194
Express ID in equation (4) with (5):
[DD = I2 - IO'F2 - (Il - Io-Fl)°Fl (6)
Express [DD in equation (3) with (6):
2-(1+F1+ 2)

R :
1.,-(1 -F,-F2) +I,-(1 +F2) +12 +F1+F2 (7)
I2 -Io-F2 -(1l —Io-Fl)-F1

 

 

When NaBD, (98% D) is used, equation (7) should be adjusted as:
R 2-(1 +1“l +172) 100
Io-(l -F1-F2) +Il-(1 +F2) +12 +F1+F2 98 (8)
I2 -Io-F2 —(Il -10-Fl)-F1

 

 

When ion pair 217/219 m/z was chosen to represent galactitol acetate/galactitol acetate-D2,
I0 = intensity of ion 217. Since Ion 217 contains 13 H, 9 C and 6 O, the abundance ratios, F l

and F 2 can be calculated as:

 

 

 

 

 

 

 

Ion I0 Il I2
NaturalDin 13H 0.01x13=0.13

13C in 9 C 1.1 x 9 = 9.9 0.006 x (9)2 = 0.486
17O in 6 O 0.04 x 6 = 0.24

18Oin60 0.2x6=1.2
Relative abundance 100 0.13 + 9.9 + 0.24 = 10.27 0.486 + 1.2 = 1.686
Abundance ratio (F) 1 Fl = 0.1027 F2 = 0.01686

 

 

 

 

 

 

Similarly, when ion pair 289/291 m/z was chosen to represent galactitol acetate/galactitol

acetate-D2, I0 = intensity of ion 289. Since Ion 289 contains 17 H, 12 C and 8 O, the

195

abundance ratios, F, and F 2 can be calculated as:

 

 

 

 

 

 

Ion I0 I, I2
NaturalDin 17H 0.01x17=0.17

13C in 12C 1.1 x 12: 13.2 0.006x(12)2=0.864
17O in 8 O 0.04 x 8 = 0.32

18Oin8O 0.2x8=l.6
Relative abundance 100 0.17 + 13.2 + 0.32 = 13.69 0.864 + 1.6 = 2.464

 

 

Abundance ratio (F)

 

 

F, = 0.1369

 

F, = 0.02464