APIDGALACTURONANS FROM THE CELL WALL 0F LEMNA MINOR L. Thesis for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY DAVID A. HART 1959 '5 my» 4 RY University THFSH‘: This is to certify that the thesis entitled APIOGALACTURONANS FROM THE CELL WALL OF LEMNA MINOR L. presented by David A . Hart has been accepted towards fulfillment of the requirements for Ph .D . degree in Biochemistry Bad/(W Major professor Date W9 é 9 0-169 BINDING BY mm; & sous lane! swam me; I Hr“ awning ‘Hlfll ABSTRACT APIOGAIACTURONANS FROM THE CELL HALL OF IEMNA MINOR L. By David A. Hart A mild, reproducible procedure has been developed for the isolation of D-apiose-containing polysaccharides from the cell wall of Lgmna‘mingg. The procedure is based on the extraction of the cell walls with 0.5% ammonium oxalate. The conditions used at 22° have no known degradative effect on polysaccharides. On a dry weight basis, the polysaccharides extracted with ammonium oxalate made up 1h¢ of the material designated cell walls and contained 20% of the D-apiose orig- inally present in the cell walls. The cell walls, as isolated, contained 83% of the D-apiose present in‘L.‘migg£. .After extraction with ammonium oxalate, purified polysaccharides were obtained by DEAE-Sephadex column chromatography and by fractional precipitation with sodium chloride. With these procedures, the material extracted at 22° could be separated into at least five polysaccharides. On a dry weight basis, two of these polysaccharides made up more than SQZ of the material extracted at 22°. There was a direct relationship between the D-apiose content of the polysaccharides and their solubility in sodium chloride solutions; those of highest D- apiose content were most soluble. All of the polysaccharides David A. Hart isolated appeared to be of one general type, namely galac- turonans to which were attached sidechains containing D-apiose. The D-apiose content of the apiogalacturonans varied from 7.9 to 38.1%. The content of esterified D-galacturonic acid resi- dues in all apiogalacturonans was low, being in the range 1.0-3.5%. Hydrolysis of a representative apiogalacturonan with dilute acid resulted in the complete removal of the D- apiose with little or no degradation of the galacturonan portion. Treatment of polysaccharide fractions with pectinase established that those of high D-apiose content and soluble in 1.0 M-sodium chloride were not degraded whereas those of low D-apiose content and insoluble in 1.0 M-sodium chloride were extensively degraded. When the D-apiose was removed from a typical pectinase-resistent polysaccharide, the remainder of the polysaccharide was readily degraded by this enzyme. Periodate oxidation of representative polysaccharide fractions and apiogalacturonans and determination of formaldehyde released, showed that about 50% of the D-apiose molecules were substituted at either the 3- or the 3'-position. The apiogalacturonans, as the sodium salts, were par- tially degraded when heated under mildly acidic conditions. The extent of the hydrolysis under these conditions was approximately equal to the percent D-apiose of the apiogalac- turonans. The same two degradation products were obtained from all of the apiogalacturonans. These were D-apiose and a disaccharide of D-apiose named apibiose. The residues from David A. Hart the degradation, the galacturonans, were not characterized. Periodate oxidation of apibiose and crystalline apibiose phenylosotriazole and determination of formaldehyde released, showed that the position of the linkage between the two D- apiose molecules was i—93'. Proton magnetic resonance Spec- trometry and molecular rotational data suggested that the linkage had the 8 configuration. Methylation analysis of apibiose and apibiose phenylosotriazole indicated that the non-reducing terminal D-apiose molecule had the D-apio-D- furanose configuration. The configuration at 0-3 of the reducing terminal D-apiose molecule was not determined. Therefore the disaccharide is O-B-D-apio-D-furanosyl-(L—93')- D-apiose. When attached as sidechains to the a-(1_9#)- galacturonan, the disaccharide is (2 and/or 3)-0-[O-B—D- apio-D-furanosyl-(L—93')-(a or B)-D-apio-(D or L)-furanosyl]- galacturonan. APIOGALACTURONANS FROM THE CELL WALL OF LEMNA MINOR L. By 1‘( \ l (\" David AI Hart A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1969 (go/M”; 3 "7'2: 7 0 Dedicated to My Wife and Parents 11 VITA The author was born, raised and educated in Marquette, Michigan. He was born in 19u2, graduated from J. D. Pierce High School in 1960 and graduated from Northern Michigan University with a B.A. degree in 1964. In the Fall of 196A he then entered graduate school at Michigan State University. After stealing food from the ducks for five years, he is presently severing ties with Lower Michigan and departing for the Windy City where he will Join the laboratory of Dr. A. Nisonoff. 111 ACKNOWLEDGMENTS The author wishes to thank Dr. Paul Kindel for his guidance during the course of this research. The author would also like to thank Dr. Loran Bieber, Dr. Clifford Pollard and Dr. Derek Lamport for serving on his guidance committee. The stimulating, lengthy, and wide-ranging discussions with fellow graduate students, in the laboratory and elsewhere, is greatly appreciated. The author is particularly grateful to his wife, Mary, for her continued encouragement and for her work as an educator of the masses to help finance this undertaking. The financial support of a National Aeronautics and Space Administration Fellowship is also gratefully acknowledged. iv ORGANIZATION OF THE THESIS The thesis is divided into three parts. Part 1 is a brief literature review of the chemistry of apiose. Parts 2 and 3 are related but deal with separate aSpects of the problem. Each of these parts has a separate Introduction, Materials and Methods, Results and Discussion. However all of the references have been placed at the end of Part 3. Part 2 has been accepted for publication in Biochem- ical Journal. Part 3 has been written in the same form as Part 2 but will be rewritten for submission to a different Journal. TABLE OF CONTENTS DEDICATION O O O O O O O O O O O O O O O O O O O VITA . ACKNOWLEmMENTS O O O O O O O O O 0 O O O O O O ORGANIZATION OF THE THESIS . . . . . . . . . . . LIS T OF TABLES O O I O O O O O O O O O O O O O 0 LIST OF LIST OF PART 1. PART 2. FIGURES O O O O O O O O O O O O O O O O SCHEPIES C O I O O O O O O O O O O O O 0 THE CHEMISTRY OF APIOSE . . . . . . . . ISOLATION AND PARTIAL CHARACTERIZATION OF AP IOGAIACTUROMNS O O O O O O O O 0 INTRODUCTION . . . . . . . . . . . . . MATERIALS AND METHODS . . . . . . . . . Materials . . . . . . . . . . . . . General Methods . . . . . . . . . . Paper Chromatography . . . . . . . . DEAE-Sephadex Column Chromatography Analysis of DEAE-Sephadex Column Fractions for Uronic Acids and D-Aplose o o o o o o o o o o o 0 Quantitative Determination of Sugars in Polysaccharide Material . . . Determination of the Content of Esterified D-Galacturonic Acid Residues in.Polysaccharide Fractions . . . . . . . . . . . . vi Page ii iii iv ix xii 11 ll 13 14 14 15 16 17 Preparation of Partially hydrolyzed Polysaccharide . . . . . . . . . . Pectinase Hydrolysis of Polysaccharide FractionS............ Periodate Oxidation of Polysaccharide Materials and Release of Formalde- hyde c c o o o o o o o o o o o o o RfiULTS O O O O O O O O O O O O O O O O Extraction and Fractionation.of L. minor 0 ,o o o o o o o o o o 0 Column Chromatography of Polysacchar- ide Fractions on DEAE-Sephadex . . Fractionation of Sodium Chloride- Insoluble Fractions with Sodium Chloride O O O O O O O O O O O 0 0 Identification of Polysaccharide Components . . . . . . . . . . . . Content of Esterified D-Galacturonic Acid Residues in.Polysaccharide Fractions . . . . . . . . . . . . Partial Hydrolysis of Polysaccharide Ila O O O O O O O O O O O O O O O Pectinase Hydrolysis of Polysaccharide Fractions . . . . . . . . . . . . Release of Formaldehyde Following Periodate Oxidation of Polysac- charide Material . . . . . . . . . DISCUSSION 0 O O O O O O O O O O I O O 0 PART 3. ISOLATION AND CHARACTERIZATION OF APIBIOSE FROM APIOGALACTURONANS . . . . INTRODLJCTION o o o o o o o o o o o o o 0 MATERIALS AND METHODS . . . . . . . . . Materials 0 o o o o o o o o o o o o 0 Plant Material . . . . . . . . . . . vii Page 18 18 19 l9 19 27 34 35 37 38 38 45 57 58 59 59 59 General Methods . . . . . . . . . . . . Chromatography . . . . . . . . . . . . Radioactive L. migg; . . . . . . . . . Isolation of Apiogalacturonans . . . . Degradation of [1&0] Apiogalacturonans Isolation of Apibiose . . . . . . . . . Sodium Borohydride Reduction of [luc] Apibiose (peak 2) , , , , , , Hydrolysis of [luc] Apibiose and Reduced.[1“C] Apibiose (peak 2) . . Preparation of Apibiose Phenylosotria- 2016 O O O O O O O O O O O O I O O O Periodate Oxidation and Determination of Formaldehyde . . . . . . . . . . Methylation.Analysis . . . . . . . . . Isolation and Pectinase ydrolysis of Partially Degraded [1 C] Apio- galacturonans . . . . . . . . . . . REULTS O O O O O O O O O O O O O O O O 0 Degradation of [1&0] Apiogalacturonans Identification of Peak 1 . . . . . . . Identification of Peak 2 . . . . . . . Chemical Characterization of Apibiose . Hydrolysis of [luc] Apibiose . . . . . Physical Characterization of Apibiose . Characterizatifin of Partially Degraded [1 C] Apiogalacturonans . . DISCUSSION 0 O O O O O O O O O O O O O O 0 APPENDIX REFERENCES Page 59 61 62 63 63 63 66 67 67 69 69 71 71 71 89 89 9o 95 95 106 106 117 118 PART 2. LIST OF TABLES Fractionation.of L. minor . . . . . . . . Efficiency of ammonium oxalate extrac- tions 0 O O 0 O O O O O O O O O O O O DEAE-Sephadex chromatography of 22° sodium chloride-soluble and insoluble fraCtlon O O O I O O O O O O O I O O O Fractionation of 22° and 70° sodium chloride-insoluble fractions with sodium chloride . . . . . . . . . . . Formaldehyde released on periodate oxida- tion of 220 extracted polysaccharides The degradation products of radioactive apiogalacturonan ZZSCS-IIa as a function of pH . . . . . . . . . . . . Periodate oxidation of apibiose and apibiose phenylosotriazole and determination of formaldehyde . . . . Chromatography of methylated D-apiose from apibiose, apibiose phenylosotri- azole and apiin . . . . . . . . . . . Summary of the proton magnetic resonance data obtained at 100 MHz for apibiose phenylosotriazole in deuterium oxide . ix Page 25 26 31 36 82 91 92 105 LIST OF FIGURES Page PART 2. 1. Column chromatogram of 22° sodium chloride soluble and insoluble fraction . . . . . 28 2. Column chromatogram of 700 sodium chloride soluble and insoluble fraction . . . . . 32 3. Column chromatogram of partially hydro- lyzed polysaccharide II-a . . . . . . . 39 4. Pectinase hydrolysis of polysaccharide fractions . . . . . . . . . . . . . . . 41 5. Pectinase hydrolysis of the 22° sodium chloride-soluble fraction before and after removal of D-apiose . . . . . . . 43 1. Scan of a paper chromatogram of thermally degraded sodium [1 C] apiogalacturonate from‘g. minor . . . . . . . . . . . . . 73 2, Degradation of [14c] apiogalacturonan ZZSCS-IIa as a function of time and temperature . . . . . . . . . . . . . . 75 3. The rate of degradation of [140] apio- galacturonan ZZSCS-IIa as a function Of pH 0 O O O O O O O O O O O O I O O O 77 4. The rate of degradation of [luC] apio- galacturonan ZZSCS-Ila as a function or pH 0 O O I O I O O O I O O O O I O O 79 5. The effect of salt cancentration on the degradation of [1 C] apiogalacturonan 22303-118 O O O O O O O O O O O O O O O 83 6. The rate of degradation of [luc] apio- galacturonan ZZSCS-IIa as a function of concentration . . . . . . . . . . . . 85 7. 10. 11. 12. The rate of degradation of [luC] apio- galacturonan ZZSCS-IIa as a function of concentration . and “'05 o o 0 Infrared Spectrum of D-apiose and api- biose . . . . Infrared Spectrum of D-apiose phenyloso- triazole and apibiose phenylosotria- 2018....- Proton magnetic resonance Spectrum of apibiose phenylosotriazole in deuterium oxide Proton magnetic resonance Spectrum of apibiose phenylosotriazole between 6.35 and 5.35 T xi . Hydrolysis of CluCJ apibiose in 0.05 M- potassium phoSphate at pH 2.5. 3.5 Page 87 93 96 98 100 103 LIST OF SCHEMES Page 1. Flow sheet outlining the procedure for fractionating L. minor cell walls . . . . 20 xii PART 1 THE CHEMISTRY OF APIOSE The Chemistry of Apiose In 1901, Vongerichten reported that an unknown sugar was released from the flavonoid glycoside apiin by mild acid hydrolysis. The sugar did not yield furfural on heating with concentrated acid. Vongerichten prepared two crystal- line derivatives of the sugar, the phenylosazone and the p-bromOphenylosazone. He concluded that the sugar was a pentose and named it apiose, after the Linnean name of the parsley plant, LpLum petroselium L. A year later, Vongerichten (1902) reported the fur- ther characterization of the apiose by preparation of apionic acid from apiose by oxidation with bromine water. This compound was converted to a crystalline strontium salt, an amorphous calcium salt and a crystalline phenylhydra- zide. Reduction of calcium apionate with hydriodic acid and phOSphorus resulted in a volatile acid which was iden- tified as isovaleric acid. He therefore concluded that apiose was a branched-chain pentose (I). In 1906, Vongerichten and Muller prepared another crystalline derivative of apiose, the a—benZyl-d-phenyl- hydrazone. Regeneration of the free sugar from this crys- talline derivative led to the first isolation of a pure syrup of apiose. Information about the stereochemistry of apiose was first obtained by Schmidt (1930). He confirmed Vongerichten's work and elucidated the configuration at 0-2 by application 2 3 of the rule for determining the configuration of the a- carbon of a-hydroxycarboxylic acids to apionic acid. He concluded that the apionic acid was D-apionic acid (II) and therefore the apiose from apiin was D-apiose (3-C- hydroxymethyl—aldehydo-D-glycero-tetrose, III). D-Apiose is unusual among pentoses because when it cyclizes it can only form a furanose ring. Furthermore, cyclization results in the formation of two new asymmetric centers at C-1 and C-3. This leads to the possibility of four cyclic isomers for D-apiose, two d-isomers (IV and V) and two B-isomers (VI and VII). In solution these are in equilibrium with a fifth isomer, the aldehydo form. The nomenclature for the cyclic isomers has not been officially established. However, it has been suggested by Cahn (in Bell, Isherwood and Hardwick, 195#) that the following nomenclature be employed. The designation "D-apio" should refer to only the configuration at 0-2. When a furanose ring is formed, the stereochemistry at C-3 Should be desig- nated with a second D- or L-, depending on whether the hydroxymethyl group at C-3 is Lgagg or 2L5, reSpectively, to the hydroxyl group at 0-2. Using such a system, the structure (IV) is designated a-D-apio-D-furanose. Only members of the D-series have been described since this is apparently the naturally occuring isomer. However, there are also four possible cyclic isomers for the L-series, for a total of eight cyclic isomers for apiose. 11 Both D-apiose (Gorin and Perlin, 1958, Khalique, 1962, and Williams and Jones, 1964) and L-apiose (Schaffer, 1959, and Weygand and Schemiechen, 1959) have been synthe- sized chemically. There have been several numbering systems employed by different authors for the carbons of apiose. In this thesis, the carbons will be numbered as shown in (VIII). Recently, the fine structure of certain naturally- occuring D-apiose containing compounds and certain deriva- tives of D-apiose has been elucidated. The results of several workers have led to the elucidation of the struc- ture of apiin. After methylation of apiin and hydrolysis, Hamming and 01118 (1953) isolated a tri-O-methyl derivative of D-apiose and 3,h,6-tri-0-methy1-D-glucose. From this data, they concluded that the linkage between the two sugars was from 0-1 of the D-apiose to C-2 of the D-gluco- pyranosyl moiety. Halyalker, Jones and Perry (1965) inves— tigated the stereochemistry at 0-1 and 0-3 of the D-apiose moiety. They also methylated apiin and isolated the result- ing tri-O-methyl-D-apiose. The compound was identical by paper, thin layer and gas-liquid chromatography, optical rotation, and infrared Spectra, to chemically synthesized 2,3,3'-tri-0-methyl-D-apio-D-furanose. Periodate oxidation data also indicated that the hydroxyl groups at 0-2 and 0-3 were in the RAE configuration. Employing the molecular rotation theory of Klyne (1950), these workers concluded 5 that the stereochemistry of the linkage between C-1 of the D-apiose and C-2 of the D-glucose was 8. Therefore apiin is 7-0-[O-B-D-apio-D-furanosyl-(1—2)—B-D-glucopyranosy1]- apigenin (IX). Hattori and Imaseki (1959) have characterized a D- apiose containing phenolic glycoside (furcatin) from Viburum furcatum Blume. From partial acid and enzymatic hydrolysis of this compound, plus periodate oxidation, they concluded that the structure of the carbohydrate portion of the molecule was O-[O-(a or B)-D-apio-(D or L)-furanosyl- (1—6)~B-D-gluc0pyranosy1]-p-vinylphenol (X). They did not determine the stereochemistry at C-3 of the D-apiose nor did they present evidence for the glycosidic linkage between D-apiose and D-glucose. Imaseki and Yamamoto (1961) have isolated an enzyme system from the same plant which Speci- fically hydrolyzes the disaccharide: aglycon linkage. The iSOpropylidene derivatives of apiose have been studied in detail. Williams and Jones (196A) have synthe— sized the 1,2:3,3'-di-0-isopropylidene derivatives of both D- and L-apiose. The L-isomer had an infrared Spectrum which was different from that obtained for the D-isomer. The authors concluded that the difference may be due to a difference in the configuration at C-3 in the two compounds, although the structures of the two were not determined. Using D-apiose obtained from acid hydrolysates of Zostera marina, Carey, Ball, and Long (1966) have shown that the C(H,0H) I HOHZC-C-CH20H OH (I) H CHZOH H H OH OH (IV) (X) List of Structures. H0 0 \‘0 I H—f-OH HOHZC — ('2 -CH20H 0H (II) H 0 H H H H OH CHZOH on (V) (I)? (2)? . o (3')C‘-?(3l l (“)C (VIII) 0 0-R' H H OH The dotted lines indicate H o \// f H-f-OH OH HOHZC-C-CH2 OH (III) HO OH H H H H0 CHZOH H 0H 0H (IX) undetermined stereo- chemistry. The non—carbohydrate components of compounds are indicated by R and R'. 7 predominant di-O-isopropylidene derivative obtained is 1,2:3,3I-di-O-iSOpropylidene-a-D-apio-L-furanose. Graded acid hydrolysis led to the isolation of the 1,2-isopro- pylidene derivative of the same furanose form. This result in no way indicates that D-apiose exists in this form in Zostera marina. Ball, Carey, Klundt, and Long (1969) have also isolated and characterized both the 1,2:3,3'-di-0- isopropylidene and the 1,2-0-isopr0pylidene of D-apio-D- furanose. Long's group have prepared derivatives of D-apiose in which the ring oxygen has been replaced with a nitrogen atom (Halford, Ball, and Long, 1969) or a sulfur atom (Halford, Ball, and Long, 1968). This group has prepared several crystalline derivatives and intermediates of the compounds described above which they have also characterized. Additional literature dealing with the chemistry and biochemistry of D-apiose is cited in the remainder of the thesis where Specific problems are discussed. PART 2 ISOLATION AND PARTIAL CHARACTERIZATION OF APIOGALACTURONANS INTRODUCTION The branched-chain aldopentose D-apiose (3-C-hydroxy- methy1-aldehydo-D-glycero-tetrose) has been identified as a constituent of a large variety of plants. In some plants it exists as a component of a flavone, isoflavone or phenolic glycoside such as apiin (Vongerichten, 1901; Nakaoki, Morita, Motosune, Hiraki and Takeuchi, 1955; Wagner and Kirmayer, i957; Rahman, 1958), lanceolarin (Malhotra, Murti and Seshadri, 1956) or furcatin (Hattori and Imaseki, 1959). However, in.Posidonia australis (Bell, Isherwood and Hardwick, 1954), LLLL2,§E, (Bacon, 1963), Zostera marina (Bacon, 1963; Williams and Jones, 1964; Ovodova, Vaskovsky and Ovodov, 1968), Lgmga gLQLg (Beck and Kandler, 1965), L. nggg (Duff, 1965; Beck and Kandler, 1965; Mendicino and Picken, 1965; Beck, 1966, 1967), g, gang (Duff, 1965), g, pacifica and ngllospadix (Ovodova g£_gl., 1968) it appears to be primarily a component of polysaccharides, In the case of g. marina (Williams and Jones, 196#; Ovodova gg,gl., 1968), 23232.3 . (Bacon, 1963), L. nggg (Duff, 1965; Mendicino and Picken, 1965; Beck, 1966, 1967), g. pacifica and.§LyL108padix (Ovodova g£_gL., 1968) a polysac- charide fraction or fractions were isolated and shown to contain D-apiose. Beck (1967) and Ovodova g£_gL., (i968) Partially characterized their polysaccharides. In a study oflL.‘ngg£ (duckweed), one of the richest kflown sources of D-apiose, Duff (1965) showed that more than 9 10 90% of the D-apiose present was not extracted by organic solvents. Extraction of the insoluble material with a succession of alkaline conditions indicated that the bound D—apiose was continuously extracted and that no single fraction contained a majority of the D-apiose. From a study of the incorporation of luCOZ into the D-apiose of ‘L. nggg and L.‘gL222 under various conditions, Beck and Kandler (1965) concluded that the D-apiose was not part of a storage material but rather a component of the cell wall. While the present study was in progress, Beck (1966, 1967) reported the isolation of two apiogalacturonans from L. 2&223 which contained 28% and 25% D-apiose, one of which contained, in addition, D-xylose and D-galactose. We are interested in the isolation of plant polysac- charides containing D-apiose in order to determine the structure of such unique polysaccharides and to study the mechanism and control of their biosynthesis. Attainment of these goals depended on the development of reproducible methods for their isolation and fractionation. In addition, the methods should result in minimal degradation of the polysaccharides. This precluded the use of alkaline extrac- tions because of the degradative effect of such conditions on polysaccharide material (Whistler and BeMiller, 1958; Neukom and Deuel, 1958). It also precluded the use of acidic conditions due to the acid lability of the apio- furanoside glycosidic linkage (Vongerichten, 1901; Mendicino 11 and Picken, 1965). Furthermore, even near neutrality, high temperatures must be used with caution since pectins may be degraded by a p-elimination reaction.(Albersheim, 1959; Albersheim, Neukom and Deuel, 1960). We therefore examined procedures which have been used to extract pectic subStances since it is known that their extraction may be facilitated by relatively mild conditions. When such conditions were applied to L. REESE cell walls, polysaccharides rich in D-apiose were solubil- ized. The present study describes the isolation and partial characterization of these polysaccharides, MATERIALS.AND METHODS Materials ‘L. Elggg was obtained from the Battle Creek River, Bellvue, Michigan. The plant material was washed exten- sively and either used immediately or stored at -200. D-Apiose was obtained from once recrystallized apiin. Crystalline apiin was isolated from.Petroselinum criSpum (parsley) seeds by the method of Gupta and Seshadri (1952) and hydrolyzed with 0.1 N-sulfuric acid for 30 min. at 100° (Vongerichten, 1901). Pure D-apiose was isolated by partition column chromatography on acid-washed Celite 535 using the procedure of Lemieux (1962). [U-iqulD-Apiose was prepared from.UDP-[U-1“C]JD- glucuronic acid (New England Nuclear Corporation, Boston, 12 Mass., U.S.A.). The procedure of Gustine and Kindel (1969) was used to convert UDP-[U-luC] D-glucuronic acid to Com- pound III. Compound III was the designation given by these workers to the D-apiose-containing compound obtained by their procedure. After paper chromatography in solvent E, Compound III was chromatographed in solvent D. For the preparation of free, radioactive D-apiose, Compound III was hydrolyzed in 0.2 N-sulfuric acid for 90 min. at 1000 and the hydrolysate treated as described previously (Gustine and Kindel, 1969). Before hydrolysis, sufficient non- radioactive D-apiose was added to give the Specific radio- activity indicated below. The free [U-luc] D-apiose from the hydrolysis was chromatographed on paper in solvents A, B and C and in each it migrated as a single radioactive peak. The Specific radioactivity was determined by measur- ing sugar using Nelson's method (19hh) with an appropriate elution blank and radioactivity using liquid scintillation counting in Bray's solution (1960). The specific radioac- tivity of the [U—l‘tc] D-apiose was 68160 disintegrations/ min./umole of D-apiose. The radioactive D-apiose was designated [U-luc] D-apiose since it was derived from the glucuronic acid portion of UDP-[U-1SC] D-glucuronic acid. Purified Fungal pectinase was purchased from Sigma Chemical Company, St. Louis, Mo., U.S.A. The enzyme was purified 30-fold over the crude preparation by the manu- facturer who stated it still contained several other enZymes. 13 The Specific activity of this batch (Lot 125B-0350) as measured by Sigma was 0.7 units per mg. of solid. A unit was defined as that amount of enzyme which liberated 1 uncle of D-galacturonic acid per min. at 25° and at pH 4.0 with de—esterified citrus pectin as the substrate. General Methods Solutions were concentrated under reduced pressure by rotatory evaporation at temperatures less than 35°. Polysaccharide material was desiccated to constant weight Ln 33329 and over phOSphorus pentoxide except in.Table 2. Calcium chloride and silver nitrate were used to test for oxalate and chloride ions, reSpectively. Centrifugations were done at 4°. Radioactivity on chromatograms was detected with a Packard radiochromatogram scanner, model 7201 (Packard Instrument Co., Downers Grove, Ill., U.S.A.). All other measurements were made in the scintillation solu- tion of Bray (1960) with a Packard Tri-Carb liquid scintil— lation counter, Model 3310. Optical rotations were deter- mined with a Zeiss Photoelectric Precision Polarimeter 0.005o (Carl Zeiss, Oberkochen, Wuerttemberg, Germany) at 22° with a polarimeter tube having a 1 cm. light path and light of 578 mu (Hg) wavelength. Polysaccharides were dis- solved in 0.067 M-potassium dihydrogen phOSphate-disodium hydrogen phOSphate, pH 7.7, unless otherwise noted, and their concentrations ranged from 9.1-21 mg./ml. 14 Paper Chromatography Descending paper chromatography was used and was carried out with Whatman No. 3MM paper prewashed with 0.1 M-citric acid followed by distilled water. The following solvents were employed: A) ethyl acetate-water-acetic acid-formic acid (18:4:3:1, by vol.), B) propan-Z-ol-water (9:1, by vol.), C) butan-l-ol-acetic acid-water (4:1:5, by vol., upper phase), D) propan—i-ol-ethyl acetate-water (7:1:2, by vol.), E) 95% aqueous ethanol-1.0 M—ammonium acetate, pH 7.5 (7:3, by vol.), F) pyridine-ethyl acetate- acetic acid-water (5:5:1:3, by vol.). Sugars were detected on chromatograms by Spraying with aniline hydrogen phthalate (Partridge, 1949) or by using the silver nitrate dip method (Trevelyan, Proctor and Harrison, 1950). DEAE—Sephadex Column Chromatography Column chromatography of the five polysaccharide fractions (see the fractionation eXperiment in the Results) was carried out on DEAE-Sephadex (A-25 medium, capacity: 3.5 m-equiv./g., 100-270 mesh, Pharmacia Fine Chemicals, Inc., Uppsala, Sweden). Before use, the DEAE-Sephadex was repeatedly suSpended in 0.067 M-potassium dihydrogen phos— phate-disodium hydrogen phOSphate buffer, pH 7.7, and the fines were removed. Before a polysaccharide fraction was applied, the column was washed with 2-3 bed volumes of the phOSphate buffer. The polysaccharide fractions were dis- solved in either water or the same buffer and applied to 15 the column at approximately the same rate as the column was to be operated. After a washing with the phoSphate buffer, the polysaccharides were eluted with a step gradient of 0.1- 0.3 M-sodium chloride in the phOSphate buffer. The indi- vidual polysaccharides were obtained by pooling the apprOp- riate fractions from the columns and dialyzing them against distilled water until negative for chloride ion. The non- diffusible material was then lyophilized and desiccated. Before characterization, some polysaccharides were converted to the hydrogen form by passage through a column of Dowex 50W-X8 (50-100 mesh, H+ form). The polysaccharide solutions were then lyophilized and desiccated. Analysis of DEAE-Sephadex Column Fractions for Uronic Acid and D-Apiose The column fractions were assayed for uronic acid by a modification of the method of Dische (1962). One-half (0.5) m1. samples or appropriate samples diluted to 0.5 ml. with water in test tubes were cooled to 4°. On top of each sample was layered 0.2 m1. of 0.1% (w/v) carbazole (recrys- tallized once from benzene) in ethanol. The tubes were cooled again to 4° and then 6.0 ml. of 15.8 M-sulfuric acid was added. The solutions were thoroughly mixed with a Vortex Jr. mixer (Scientific Industries, Queens Village, N.Y., U.S.A.) at 22°, heated at 1000 for 20 min., cooled in Water to 22° and the extinction at 525 mu determined immedi- ‘ately. With these conditions L-arabinose, D-xylose, and 16 D-apiose gave 3.4%, 4.8%, and 2.4% reSpectively of the color obtained with D-galacturonic acid. In the above procedure, the samples were heated after the addition of carbazole. This is in contrast to the original Dische procedure and has two advantages; first, the sensitivity of the method is increased, and second, the extinction readings can be made immediately after the 20 min. heating period (Gauthier and Kenyon, 1966). Early in this work it was discovered that the 22° and 70° sodium chloride-soluble and -insoluble polysaccharide fractions quantitatively released their D-apiose on mild acid hydrolysis without the release of significant amounts of other reducing material. This property was used to develop the following assay for D-apiose in the column frac- tions. A 1.0 m1. sample or an apprOpriate aliquot diluted to 1.0 ml. with water was mixed with 0.1 ml. of 1.0 N- hydrochloric acid and heated at 1000 for 30 min. The samples were cooled and the liberated reducing material was determined by the method of Nelson (1944). The extinctions were read at 540 mu. Quantitative Determination of Sugars in.Polysaccharide Material The percent D-galacturonic acid was determined, after saponification (McComb and McCready, 1952), by the above described modification of the sulfuric acid-carbazole method. D-Apiose was determined by isotope dilution. For 17 each determination, approximately 5-10 mg. of polysaccharide material was suspended in 1.0 ml. of [U-14C] D-apiose con- taining 9474 disintegrations/min. and 1.0 ml. of 1 N-sulfuric acid was added. The solution was heated at 1000 for 1 hr.. cooled to 22°, neutralized to pH 6-7 with sodium hydroxide, and then nine volumes of absolute ethanol was added. The resulting precipitate was removed by centrifugation. The supernatant solution was decanted, concentrated, streaked on paper and the chromatogram was deveIOped in solvent B. A strip of the chromatogram was treated by the AgN03 dip method. The material which cochromatographed with the radioactive D-apiose was eluted and restreaked on paper and the chromatogram developed in solvent C. After elution, the Specific radioactivity of the radioactive D-apiose was determined by measuring reducing sugar and radioactivity as described above. The amount of D-apiose released from the polysaccharide material was calculated from the percentage of the total radioactive [U-140:]D-apiose recovered and the Specific radioactivity of the isolated and added [11—140] D- apiose. Determination of the Content of Esterified D-Galacturonic Acid Residues in.Polysaccharide Fractions The methoxyl content of polysaccharide fractions was determined by the method of Schultz (1965). Polysaccharide fractions (20-50 mg.) were dissolved in carbon dioxide-free water, titrated to a phenolphthalein endpoint with 0.005 18 N-carbonate-free sodium hydroxide, and then 5-10 ml. of 0.1 N-carbonate-free sodium hydroxide was added. The solutions were kept at 22° for 30 min. and then an equal volume of 0.1 N-hydrochloric acid was added. The solu- tions were titrated to a phenolphthalein endpoint under nitrogen with the 0.005 N-sodium hydroxide. With this method, a value of 8.3% was obtained for the methoxyl content of citrus pectin (grade II, Sigma Chemical Company). The percent of esterified D-galacturonic acid resi- dues in the polysaccharide fractions was calculated from the percent D-galacturonic acid, obtained from the sulfuric acid-carbazole test, and the methoxyl content obtained as described above. Preparation of Partially Hydrolyzed Polysaccharide D—Apiose was removed from the 22° sodium chloride— soluble fraction by hydrolysis with 0.1 N-hydrochloric acid for 30 min. at 100°. The solution was cooled, neutralized with sodium hydroxide to pH 7, and then dialyzed against distilled water until the diffusate was negative for chloride ion. The non-diffusible material was lyophilized and stored over phoSphorus pentoxide Lg yagug. This material was used in the pectinase eXperiment depicted in Figure 5. Pectinase Hydrolysis of Polysaccharide Fractions Polysaccharide fractions were incubated with pectinase at 37° in 0.025 M-sodium acetate buffer, pH 4.5. The enzyme 19 was dissolved in 0.05 M-sodium acetate buffer, pH 4.5. and the polysaccharide fractions were dissolved in distilled water. The reaction was followed by measuring the increase in reducing material. as determined by the method of Nelson (1944), with time. The extinctions were read at 540 mu. Periodate Oxidation of Polysaccharide Materials and Release of Formaldehyde Polysaccharide material (approx. 5.0 mg.) was dis- solved in 1.0 ml. of 0.05 M-sodium acetate buffer, pH 5.0, and then 1.0 ml. of 0.1 M-sodium metaperiodate was added. The solution was kept at 22° for 2 hr. in the dark. Pre- liminary eXperiments showed that the release of formalde- hyde was virtually complete after 1 hr. Formaldehyde was determined using chromotropic acid (Speck, 1962). The chromotrOpic acid was recrystallized once from aqueous 50% (v/v) ethanol. RESULTS Extraction and Fractionation of'L. minor The'L.‘ngg£ plants were initially extracted so that cell walls were isolated. The cell walls were fractionated by the procedure outlined in Scheme 1. A typical fraction- ation eXperiment is described below. Fresh.L..nggg (350 g., 24.15 g. dry weight) was homogenized for 1 min. periods in a Waring Blendor succes- sively with 1.0 M-sodium chloride (3 times), water (1 time), 20 .wHHmz Haoo Honda .m_waapmSoapomhu you oasoooona on» deSaHSSo pooch roam .H madman 21 :oapodhu cansdonca noduocuu cansaon n.6«HOH80 flfldcom och I06uh0d£o lfiavom 005 coduoauu sansaou:« nonauoano season o- noduoauu oHnSHou loudhoazo Eflduom ONN seasonaaasaohq soda-uaaanaonq «gonadan uaanadaa oududndoenm undudnuonsm. nodudwuuduunoo H0121: o.~ non» .oa.»flda Asa-onset ouuuandooum unduunkonsm SeduIBSuuhpnoo cadaewo ladsoaid un.o as eosfionuaa Renae-ac. ouduanaoekm vacuucuonsm ScavdMSuauvneo od«uoa:o idaoado now onus-om nouauuaaa oocapaoo _ _ ooa .noaa: .m ooa .ounauwo asasoaa< um.o .« osouuom _ :oauanaaanaonq soapsnaaa:Aoaq nanhaldn muahaeda ouuaandoohm acuudchonam r'l'lll. coauam=uahunoo HUHZIS o.~ con» .wamhaedn “Upcomacv ouuvdaaooum ucmpdnuoasm codammauauuSoo ouuauwo adanoaau um.o a“ so>aonuaa thuonuu. oueuaaaooum ucuuacuoaam noduemsuahuCov opahoaSo asdoflao Rea moudkufldh vocanaoo . owm .uovm: ”N ONN oudeNo fiascoaad mm o a escauom — ceapomuu Hope: con aoauouaaazaoaq nopdauaaa escapaoo . Hope» .u: SN .ooa-oo .hopa: mafia! HHoo 22 0.1 M-sodium chloride (2 times), and water (3 times), using 1 l. quantities for each homogenization. The suSpension was filtered through six layers of cheesecloth after each homogenization. The final residue was washed with water until the washings were free of chloride ion, dried by solvent exchange with aqueous 95% (v/v) ethanol, absolute ethanol, absolute ethanol-diethyl ether and diethyl ether and desiccated to yield 7.97 g. of white material, desig- nated cell walls. Cell walls, 7.50 g., were suSpended in 1 l. of water and stirred at 60-700 for 8 hr. The suSpension was filtered by suction and the process was repeated twice. The combined filtrates were concentrated, lyophilized, and desiccated to yield 280.6 mg. of brown material. This was called the 70° water fraction. The residue was suSpended in 1 l. of 0.5% (w/v) ammonium oxalate, pH 6.2, and stirred at 22° for 3 hr. The suSpension was filtered and the process was repeated. The residue was then extracted with 1 l. of water at 22° for 3 hr. and the suSpension filtered. The extraction of the residue with water was repeated once and the four filtrates were combined and concentrated to approximately 400 ml. The solution was dialyzed against distilled water until the diffusate was negative for oxalate ion. The solution of non-diffusible material was transferred to a beaker and 10% (w/v) calcium chloride was added slowly to the stirred 23 solution until no further precipitation occurred. After cooling to 4°, the suSpension was centrifuged at 35000 x g. for 15 min. and the supernatant solution was decanted and discarded. With stirring, the precipitate was redissolved in approximately 400 ml. of 0.5% (w/v) ammonium oxalate at 22°. After cooling to 4°, the insoluble calcium oxalate was removed by centrifugation at 35000 x g. for 20 min. and discarded. After decanting, the supernatant solution was dialyzed against distilled water until the diffusate was negative for oxalate ion. The solution of non-diffus- ible material was concentrated to 300 ml. and an equal volume of 2.0 M-sodium chloride was added dropwise to the constantly stirred solution. After cooling to 4°, the suSpension was centrifuged at 35000 x g. for 15 min. The supernatant solution was concentrated to about 250 ml. and then dialyzed against distilled water until the diffusate was negative for chloride ion. No further precipitation of polysaccharides occurred during this reduction in volume even though the concentration of the sodium chloride in the supernatant solution increased to at least 2 M. The non- diffusible material was lyophilized and desiccated to yield 371 mg. of white material. This material was designated the 220 sodium chloride-soluble fraction. The material precipi- tated by the 1.0 M-Sodium chloride was resuSpended in water and dialyzed against distilled water until the diffusate was negative for chloride ion. The solution of non-diffusible material was lyophilized and desiccated to yield 321 mg. of 24 white material. This material was designated the 22° sodium chloride-insoluble fraction. The residue from the 22° extraction procedure was further extracted with 1 l. of 0.5% (w/v) ammonium oxalate. pH 6.2, at 700 for 3 hr. The suSpension was filtered and the process was repeated. The residue was then extracted with 1 l. of water at 70° for 3 hr. and the suSpension filtered. The extraction of the residue with water was repeated once and the four filtrates were combined. The material solubilized by the 70° extraction procedure was taken through the same steps as that solubilized by the 22° extraction procedure. The material designated the 700 sodium chloride-soluble fraction weighed 211 mg. and was yellow, while that designated the 70° sodium chloride- insoluble fraction weighed 105 mg. and was white. The residue from the 70° extraction was dried by solvent exchange as described above for the cell walls. It was then desiccated to yield 5.15 g. of material. The results of this fractionation eXperiment are summarized in.Table 1. In a separate eXperiment, the efficiency of the individual ammonium oxalate and subsequent water extrac- tions was examined. The results are Shown in Table 2. 25 .mHHms HHoo poapp on» mo pneumoo omofiamim on» no mammp onp co bopmHSono mosHm>+ .mpcman poano on» no pneumoo ow0aamim oz» mo mammp on» so poudadoamo mosam>t a.oa m.mm ~.ou m.mo m.oa o.mo as.m osoamom m.a H.a o.sa s.a Naa.o oHpsHoosauooaaoHso season ~.s m.m o.am m.m smm.o oHosHoo-ooaaoH:o asaoom Soduomapwo opmHmNo anasoaad oon o.m m.m H.0a m.s Ham.o oaosaoosauooaaoaso ssaoom m.aa m.d a.mm o.m mam.o oaosaomuooaaoaso asaoom COHUOGHUNm OPMHQNO BfidflOE—HQ CNN a.o a.o . a.m a.m aa~.o hoses cos ooH a.mm h.¢a ooa no.5 Sass HHoo 1.. cos m.m -1- ma.sm mossao and +Amv *Amv ARV ARV A.wv Soapomum some :« Uohobooon woodedrn pampcoo woodedia bondmpno psaoa< sodpooam .popmcoapomnw mm: Hmanopma Hams Haoo mo .w mm.m pas» mammn on» no oopmHSOHmo Soon obs: made: aaoo emu Bonn nonadpno mcoapomnm Sam on» mo mpnwaos one nonas .m ho Soapmcodpomam .H wands 26 Table 2. Efficiency of the ammonium oxalate extractions ‘L.‘mL22£ cell walls were extracted with water at 70° as described in the text and dried. This material (3.8 g.) was further extracted as described in the text. For each extraction, 400 ml. of extrac- tant was used. The individual extracts were concen- trated at 30°, dialyzed against distilled water, lyOphilized and weighed. Extractant Amount extracted ms. % 0.5% Ammonium oxalate, 22° 335.1 8.8 0.5% Ammonium oxalate, 22° 24.4 0.6 Water, 22° 18.8 0.5 Water, 22° 11.3 0.3 0.5% Ammonium oxalate, 700 89.2 2.4 0.5% Ammonium oxalate, 70° 26.2 0.7 Water, 70° 27.5 0.7 Water, 70° 2.1 0.1 Residue 3200 84.2 27 Column Chromatograpgy of Polysaccharidg Fractions on DEAE-Sephadex The five fractions obtained from the fractionation experiment described in the preceding section were sub- Jected to column chromatography on DEAE-Sephadex. The procedure described in the Materials and Methods section was followed. The 70° water fraction (110 mg.) was suSpended in 35 ml. of water and stirred several hours, first at room temperature and then at 60-700. Since all the material did not dissolve, the suSpension was centrifuged at 35000 x g. for 15 min. The supernatant solution was decanted and the precipitate was dried with acetone and weighed (13.2 mg.). The material in the supernatant solution (96.8 mg.) was applied to a DEAE-Sephadex column (2.2 cm. i.d. x 25 cm.). Fractions were collected at a rate of 0.20 ml./min. No material was eluted which reacted in the tests for uronic acid and D-apiose. The top of the column contained a yellow band which did not elute. Because of its low D-apiose content, further attempts to characterize this fraction were not made. The 22° sodium chloride-soluble fraction (335.2 mg.) was dissolved in 20 ml. of the phoSphate buffer and chro- matographed on a DEAE-Sephadex column. The elution profile is given in.Figure 1a. The indicated fractions were pooled and the polysaccharides were isolated as described in the 28 Figures 1a and 1b. Column chromatograms of 22° sodium chloride-soluble and -insoluble fractions. Figures 1a and 1b are the elution profiles for the 22° sodium chloride-soluble and -insoluble fractions, reSpectively. The columns (both 2.8 cm. i.d. x 25 cm.) were of DEAE-Sephadex and were develOped identically. The columns were treated with the phoSphate buffer from fractions 17-39. For both columns, 15 ml. fractions were collected at a rate of 0.3 ml./min. The fractions were assayed for uronic acid (0) and D-apiose (A) as described in the Materials and Methods. 29 0.3 M NoCl L 0.25 M NOCI 0.2 M NOCI 4. A3 cozoo: we 7: ton oeom _ _ Ib a w ? IZO I30 Fraction number _ m TV cozoot So _E 58 owow A3 cozoot Co 2: Lou oeom 3 05 5 A (b) l5)— _ m 2 A m 0 mar L l' _ 5 l20 ISO Fraction number 75 95 5 5 is TWO 0 A: cozoot yo .8 son anew 30 Materials and Methods section. The analytical results are summarized in Table 3a. 3 The 22° sodium chloride-insoluble fraction (285.5 mg.) was dissolved in 40 ml. of the phOSphate buffer and chromatographed on a DEAE-Sephadex column. The elution profile is given in Figure 1b. Additional washing with 0.5 N-sodium hydroxide did not result in the elution of more polysaccharide material. The indicated fractions were pooled and the polysaccharides were isolated. The analyt- ical results are summarized in Table 3b. The 70° sodium chloride-soluble fraction (190 mg.) was dissolved in 30 ml. of the phOSphate buffer and chro- matographed on a DEAE-Sephadex column. The elution pro- file is given in Figure 2a. Additional washing of this column with 0.5 N-sodium hydroxide did not result in the elution of more polysaccharide material. The indicated fractions were pooled and the polysaccharides were isolated. On a weight basis, the amount of material recovered as polysaccharides Ia, Ib, 11a, 11b and III was 9.8, 6.4, 28.8, 15.3, and 5.1%, reSpectively. of that applied to the column. Only polysaccharide IIa was analyzed. In the hydrogen form, it contained 34.1% D-apiose and 52.3% D- 22 578 The 70° sodium chloride-insoluble fraction (91.0 mg.) galacturonic acid and had [a] + 107.1°. was suSpended in 40 ml. of water and stirred for several hours. Since all the material did not dissolve, the sus— pension was centrifuged. The supernatant solution was 31 O O N m o.mm m.aa 0.: o.sH nHHH s.mm m.ma m.oa s.mm sHHH H.mm m.om m.ma o.mm HH .1 1: m.m o.oH H Sodpomhm oHDSHomcaiopanoano Baapom omm ADV o.mm I. I. I fin SS HS 11 in 11 m.m s.mH nHH oa.moa+ m.mm H.m 5.0m m.ama mHH om.oa+ H.ms a.mm m.om m.mm pH om.Hm+ m.ba H.0m 0.:H 0.0: 8H Soduomhm oHnsHomiolooaso azauom oNN Adv ARV Amy “av A.mav N. Gaow QESHOO SOL...“ mmmmau oaSoHSpomHmcia omoHQSIQ donoboooh unsoaw ovanmsoommhaom .modpdmoa Hmmoomaw mm commoaaxo Show +m Hams» SH mopanmnoommmaoa means on» mo mowmpnooaoa one mosam> Amman one .Soapmndnop nomnmso onomon pad» on» SA dondaomoo mm Show +m on» op nu popaobnoo ohms moss .muamm asdpom adonp mm ooswaoz one: sasaoo on» aoau modanmsoommmaoa one msoapomam oHQSHomSHI can mansaomuopaaoano adupom omm mo mnamamopmaoazo Noomsmomlmqmn .m wands 32 Figures 2a and 2b. Column chromatograms of 70° sodium chloride-soluble and -insoluble fractions. Figures 2a and 2b are the elution profiles for the 70° sodium chloride-soluble and -insoluble fractions, respectively. The columns (both 2.2 cm. i.d. x 25 cm.) were of DEAE-Sephadex and were deveIOped identically. The columns were treated with the phoSphate buffer from fractions 1-10 and with 0.1 M-Sodium chloride in phOSphate buffer from fractions 11-29. For both columns, 15 ml. fractions were collected at a rate of 0.25 ml./ min. The fractions were assayed for uronic acid (0) and D- apiose uh) as described in the Materials and Methods. per ml of fraction (0) 5525 5525 Per ml of fraction (0) 33 L 0.2 M NaCl 0.25 M NaCl I 0.3 M NaCl r r ‘T , (a) 7.5— q -« I5 10 lb 110 no In a s a e e 4 >———-—————l 5.0— if“, .6“ . " Fraction number ,I' I :\ Z .'.\. ()qu ALL—“31:111., AmmL/ ABM W ' O O 30 40 60 75 IOO llO l35 Fraction number b 7.5.- ( I .i 1.5 50»— in ma me d '0 a? —4 v——+———-—-l 2.5L— .. ~05 "3. 1"! \..\ N" -.. l O A—ma—n—AqLA-A-AL' “ISIWAIO OLSLOI'ZIO 60 75 IOO llO |35 E540 per ml of fraction (A) E540 per ml of fraction (A) 34 decanted and the precipitate was dried with acetone and weighed (9.9 mg.). The material in the supernatant solu- tion (81.1 mg.) was chromatographed on a DEAE-Sephadex column. The elution profile is given in Figure 2b. Additional washing of the column with 0.5 N-sodium hydrox- ide did not result in the elution of more polysaccharide material. The indicated fractions were pooled and the polysaccharides were isolated. On a weight basis, the amount of material recovered as polysaccharides II, 111a, and IIIb was 20.6, 17.3, and 33.8%, reSpectively. of that applied to the column. No further work was done with these polysaccharides. Fractionation of Sodium Chloride-Insoluble Fractions with Sodium Chloride In addition to fractionation on a DEAE-Sephadex column, the 22° and 70° sodium chloride-insoluble fractions were also fractionated with sodium chloride. The 22° sodium chloride-insoluble fraction from a different preparation (500 mg.) was dissolved in 100 m1. of water and 2.0 M-sodium chloride was added dropwise slowly to the constantly stirred solution until the desired molar- ities were reached. Precipitated polysaccharides were removed at 0.27 M-, 0.41 M- and 1.0 M-Sodium chloride con- centrations by centrifugation. Centrifugation was at 35000 x g. for 15 min. Increasing the sodium chloride concentra- tion to 2.0 M by concentrating the supernatant solution from 35 the final centrifugation did not result in further precipi- tation. The precipitated polysaccharides were suSpended in water and these suSpensions and the supernatant solution were dialyzed until negative for chloride ion and then lyophilized. After desiccation, the polysaccharides were characterized. The results are summarized in Table 4a. The 700 sodium chloride-insoluble fraction (200 mg.) was dissolved in 40 ml. of water and 2.0 M-sodium chloride was added as above. Precipitated polysaccharides were removed at 0.41 M- and 1.0 M-Sodium chloride concentrations by centrifugation. Increasing the Sodium chloride concen- tration to 2.0 M as described above did not result in fur- ther precipitation. The polysaccharides from the precipi- tated fractions and the supernatant solution were isolated as described above and characterized. The results are summarized in Table 4b. Identification of Polysaccharide Components The procedure used to determine the percent D-apiose content of the polysaccharide fractions in Table 1 also served to identify D-apiose as a component of these frac- tions. Solvents B and C distinguish between D-apiose, L-rhamnose, and Ipfucose. The identification of D-galacturonic acid as a com- ponent of the sodium chloride-Soluble and -insoluble frac- tions was based on the following. All four fractions reacted positively in the sulfuric acid-carbazole test. 36 O\ o (I) O\ III o.Hd m.mm 0.5 m.ma 1:1 pampHSHomsw Q om.maa+ m.om m.aa a.aa m.mm esoasoooam oo.auma.o o oH.NmH+ a.sm m.n o.mm a.asH Hoe as.ouum.o m soaeoaaa mapsaonsauooaaoaso asaeom ooa any o.~a om.aaa+ m.mm n.2N 0.“ m.mm all psapaahoasm Q oa.oma+ m.mm N.aa m.aa ~.am esoasoooam oo.a-ma.o o om.mmm+ 3.0m «.0 o.am o.mmm Hos as.ouam.o m om.on+ m.aa m.a a.m m.am esoasoooam am.ouda.o < soaoosaa oaosaonsauooaaoaao season omm Add may Ame ARV A.msv Lav mum odoa poaoboooa opapaaaooaa Hoaz mo mmmdu oHSoaSpomHmoiQ encaadim pssoaw mo mama .aosoo opanmnooammaom on» you nouns poaadpmap SH pmbaommab one; womanmSoommmHoa one .mpSoaoHSmmoa Soapmpoalaaoapao .mosodmoa ammoomaw ma commoaaxo Show +mz on» ad mopapmsoommzaoa define 0:» mo mowapaoopoa ohm mosHab Human ass azaoom and: mzoapoaam oHQSHomSalothoaso asdpom cow was oNN mo SodpaSoHpomhm .mpaam adapom Hams» ma poudhopoaamso cam poswaos ohms mopananooamafloa one ooaaoaso .s oases 37 The insoluble fractions, and the soluble fractions after mild acid hydrolysis, were degraded in the presence of pectinase. Paper chromatography of the enzymatic hydro- lysates of the insoluble fractions in solvent A revealed that D-galacturonic acid was the predominant sugar present. Finally, paper chromatography of representative polysac- charide fractions in solvents A and F following acid hydrolysis under reflux for 15 hr., showed that D-galac- turonic acid was the major sugar present. These solvents distinguish between D-galacturonic and D-glucuronic acid. The D-galacturonic acid was further distinguished from D-glucuronic acid by its inability to form a lactone. In the eXperiments for determining the D-apiose content of polysaccharide fractions we normally found a single Spot on the chromatograms after chromatography with solvent B and it correSponded to D-apiose. Occasionally one and rarely two faint Spots were present also. These were not identified. These results suggest that either other sugars are not present at all or only in small amounts in these polysaccharide fractions or there was incomplete hydrolysis. Content of Esterified D-Galacturonic Acid Residues in.Polysaccharide Fractions The percent of esterified D-galacturonic acid resi- dues in the 22° and 70° sodium chloride-soluble and -insoluble fractions was determined. The values for all 38 four fractions were similar, ranging from 1.0-3.5% with most values between 2.5 and 3.5%. Partial Acid Hydrolysis of Polysaccharide 11a The results of the partial acid hydrolysis and rechromatography on DEAR—Sephadex of 22° sodium chloride- soluble polysaccharide IIa (see Figure 1a) are Shown in Figure 3. Paper chromatography in solvents A and B of aliquots of the column fractions containing reducing material revealed that D-apiose was the only sugar present. No reducing material was associated with the uronic acid- positive material. The uronic acid-positive material that was eluted correSponded to about 30% of theory and was characterized as a galacturonan by its lack of reactivity in the Nelson test and by its conversion to D-galacturonic acid on treatment with pectinase. No additional D-apiose was released on further acid hydrolysis of the eluted galacturonan. Pectinase Hydrolysis of Polysaccharide Fractions The results of the pectinase hydrolysis of the sodium chloride-soluble and -insoluble fractions are shown in Figures 4 and 5. From Figure 4 it can be seen that only the sodium chloride-insoluble fractions were extensively degraded by the pectinase treatment. Paper chromatography of the pectinase hydrolysates of these two fractions in solvent A revealed that the reducing material was predom- inantly D-galacturonic acid. The maximum reducing values 9 3 .mUOSpmz 0:0 mamanmpmz 0:» Cd vanahomom mm 33 omoamaim 0cm ADV 30m 320.? no.“ command one: can .SHE\.HB 04.0 mo mama a pm mopooafloo mums A.Ha 0.0V mcodpompm .ammmsn mama on» ad opaaoaso azapomiz m.01H.0 mo pcoapanw mopm a Spas comp 0am .m.m ma .Hommsp opasamosa nowoapms esdpom lopmnamosa mowOHUmnap anammmpoaia $00.0 Spas pmaam popsao was madaoo one .A.ao 0m x .0.“ .So :.Hv Sasaoo Noumnaomlmmmm m on podamaa 0am opaxoapms anamom Spas moNHHSHSSoS was :oHpSHom one .000H pa .Saa on now paoa oaaoasooapms z H.0 SH poumaoaoms was A.wa mmv mHH spasmsoommmaoa mansaomloUHHOHSO azauom omm one .aHH opaaazoommmaoa moumaoapmn maaaapaaa 0o anamopaaoaso sasaoo .m oaswam (v) uoglooii 101w 18d 0’93 Q N 0. v 13:5: c282“. L. .002 .2 nd oz 2 8.0— 63 .2 «0+ Go: 5 _.o._I ON 0.? 9293 (e) uogioaii )0 ILU Jed 41 .codpomnm oHDSHowcdlopdhoano anapom oou .AO; “modpoanm oHDSHomlopaaoaso asduom oon .500 “nodpomhh mansaom2a100Hhoaso adauom omm .Ads "Soapoahm oapzaomiopaaoaso addoom oNN .Ads "one: ponmaoapms maodpomhm opaamsoomwmaoa one .pospoa haemav Somaoz on» an doaaaaopop was Hmanopaa wzaospom .Honpowop ponmaaam HHS pan» Seam popaapm who; maoHSHQSOSH .Soapsaom omaaapooa on» ohomop 0000s was anomaoa Hoaaoo oSameHm cowaoz on» paooxo mops» oaaamm amp ad ad mama amp was mobs» SoapSDSOSA on» on mamdaopms mo Sodpaoum no Macao on» .maoapa950sd asap onou on» SH .000H pa madame: an maopadpoaaa UoSoHHom mpcowmoh Somaoz 0:» mo Soapappa as was Sodpmmaanop Soapomom .omaaapooa mo .wn 00w 0am Soap soapy opahmsoommmaoa as» no pawn asapom on» no .m: 00m coaaapnoo onbpxaa mammm 20mm .oasHo> Hazam .Ha 0.H mo agapwaa gamma oSo mpaomohaoa paaoa Seam .maoapoaam opaaasooammaoa 0o mammaonpms ommcapoom .: oaswdm 42 as: as: oo 1:. oo om 8 o. oo Iago llllllllllllll Oi llllll QUINN HO O 1. \2 \\\G \G \ \0x [I “““ II. 1.10.— II‘\\ lllllllllll \q j I _ll\\i _ — _ _ m._ Obga 43 .pospoa Asamav Somaoz map an monaahopop mas Hmaampaa madcapom .pozoaaou was : ouswam 00 bummed on» ad canaaommo ohdpoooaa Hangmaanoaxo one .omaSHpooa no .wj cow was .mpamm azapom on» ma anon .Soapoaam opaamSooammHoa poumaoapmx mo .wj 0NH Ho pouzaoapmnss mo .mn 00m nonpao pmaampaoo oHSSHHa manna seam .oasHob Hanaw .Ha 0.H mo unapxaa manna 0:0 mpaommaaon paaom seam .omoHaalo no Hmpoaoa AOV Hopmm 0am ADV apogee. Sodpoahm oHDSHomlopdaoHno adapom omm on» mo mamzaoapmn omaaapoom .m oaswam sea 8.: 0m . 0a on em 0. 17 _ _ _ _ 0 1 _ .l 0.1 \ I I. 30.30 5.3 5:00: Staggered I me I m0 ||.I\\O 323-0 SSE! c289; 02.6503on _ _ _ _ 0993 45 attained by the insoluble fractions correSpond to a D- galacturonic acid release of about 80% of theory. The sodium chloride-soluble fractions were resistant to pectin- ase-catalyzed hydrolysis. However, incubation of such a polysaccharide fraction with pectinase after removal of the D-apiose by dilute acid hydrolysis resulted in exten- sive degradation, as shown in Figure 5. The maximum reducing value attained with the hydrolyzed 220 sodium chloride-soluble fraction correSponds to a D-galacturonic acid release of about 60% of theory. Release of Formaldehyde Followingwgeriodate Oxidation of Polysaccharide Material The 22° sodium chloride-soluble and -insoluble frac- tions were oxidized with sodium metaperiodate and the amount of formaldehyde released was measured. In addition, certain of the polysaccharides obtained following separation of the 22° sodium chloride-soluble fraction on DEAE-Sephadex were also oxidized. The 220 sodium chloride-soluble polysacchar- ide IIa (see Figure 1a) was treated with sodium metaperiodate both before and after it was subjected to a hydrolysis pro- cedure which liberated only D-apiose. These results are summarized in Table 5. 46 .mphnopamaaom 0o oasooaoa oao panama» vacancy omodamia oao pan» mamap amp no copaHSono was mpmsopamaaom no paoda Hmo lapohomnp one .coppm was .0.m ma .poCMSD opapoom azapomlm m0.0 no oBsHop Hasvo am can ooauoano asapomuz H.o 0o .HE 0.H ad pmpaowmao was medaa£oommmaoa ponmaoapmnas one .0oaaomaoa mm: Sodpmpaxo opmpoahoa on» use momma was .0.m ma .aommsn opmpoom adapomiz m0.0 mo .Ha 0.H cone .00008 was opaxoapms adapomlz H.0 0o oasaob Haddo mm was poaooo was Soapsaom one .Ufioa canoanooapmniz H.0 0o .HB m.0 SH o00H um .naa on How opdamnooawmaoa was» madame: an pandaoam was aHH oHDSHom oNN momma Ichpmm .Aaa onsmam oomv moapomhm mansaomlopaaoamo azapom omm on» go asamnwopma loano sasHoo wovasaomlmmmm Bonn pocaauno mmpaamnoommmaoa amp op Momma aHH 0am DH .mH mansaom omm one .mam>apooamoa .maoapomam mandaomSHi 0cm oHQSHomlopaaoano season omm man on amass mapsaon omm ass mansaomsa omm .oaome on» sH .moosaoz 0am mHaHaopmz on» ad popaaomoo mm poaaoaaoa who: msoapmpdxo mumpodnom mopahazo nommmHoa popomhpxo omm mo moapmpawo opmpoahoa mo mommoaoa opmnovaaahom .m manna 1+7 opaaazooamaaoa u .maoms m.ma m.om m.mm m.Hm m.mm H.mm o.mm mam.m HNO.H mam.a Hom.a Hum.a mma.a mmm.o omm.m 0mm.m somm.~ 0mm.m mma.m omm.~ ssm.o 0wm.m 0mm.m omm.m omn.m mms.m omm.m sam.0 poumaoapmm poumaonphsso sHH oaosaom omm mHH pH mH oaasaom omm oaasaonaH omm . o c e o o moan mucosa mo R .aaoa .wa\moHo83 maop wa\moHoan ..maoa wa\m H uasoh dopooaxo sand» ooasooadsaom opanouamahom owodadlm aaaamm 48 DISCUSSION The procedure develOped for the isolation of D- apiose-containing polysaccharides from the cell wall of ‘L..mL§gg is simple, mild and reproducible. The conditions used at 22° have no known degradative effect on these polysaccharides. The procedure is reasonably quantitative since 86% of the starting cell wall material and 97% of the D-apiose of the cell wall could be accounted for in the various fractions. With this procedure, the major apiogalacturonans can be obtained in sufficient quantity to permit structure determination. We established that the polysaccharides isolated were components of the cell wall by isolating cell walls before any fractionations were performed. An extensive series of extractions was used in the preparation of the cell walls to minimize possible contamination from other cellular components. Although this resulted in the solubilization of two-thirds of the plant material, 83% of the D-apiose was still present in the final product, designated cell walls. This is a mini- mum value since some of the cell wall material may have been solubilized by the shearing action of the blendor and some may have been lost in the cheesecloth during the fil- trations. The cell walls were not further characterized to determine purity. The results clearly Show that, at least inlL.‘nggg, most of the D-apiose present in this plant is in cell wall components. Its function there is unknown. 1.9 The polysaccharides isolated with our procedure belong to the pectic acid group of plant cell wall poly- saccharides. The degree of esterification of the D-galac- turonic acid in all polysaccharide fractions was very low. Similar results have been reported by Beck (1967). This appears to be their natural condition since the isolation procedure does not contain any steps which would lead to de—esterification. Highly esterified polysaccharides, if present in these cell walls, were probably lost during the Preparation of the cell walls. Cold water-soluble poly- saccharides, in general, would be lost during the isolation of cell walls by our procedure. Because of this, the weight Value for the 70° water fraction in Table 1 is a minimum one. The results in Table 1 show that on a dry weight basis, the four polysaccharide fractions extracted with aJllrlionium oxalate made up 14% of the cell walls and con- talned 20% of the D-apiose originally present in these cell WeI-Zl.ls. They were presumably non-covalently bound in the Be 11 wall, possibly as calcium salts, since they were T~‘eadily extracted with ammonium oxalate. A portion of the aipiogalacturonans could only be extracted by 70° ammonium oxalate. All of our experiments showed this material to be similar to that extracted at 22° but it may be partially degraded due to the temperature. At 1.0 M—sodium chloride, the material extracted by 50 ammonium oxalate at 22° was separated into two fractions with strikingly different D-apiose contents, 33.1% versus 10.1%. Similar, though not so striking, results were obtained with the material extracted at 70°. Although the 22° sodium chloride-soluble fraction separated on DEAE- Sephadex into two or possibly three polysaccharides, the differences between these polysaccharides were slight and may represent natural variations within one type of poly- saccharide or possibly some degradation of one type of polysaccharide. The DEAR-Sephadex chromatography of the 22° sodium chloride-insoluble fraction was not entirely successful. The yields were low and the resolution was incomplete. However, sufficient information was obtained to Show that it was a mixture of partially separable com- ponents. The chemical and physical properties of the polysaccharides obtained from the 22° sodium chloride- insoluble fraction are different from those of the 22° sodium chloride-soluble fraction. The elution profiles of these two fractions show that polysaccharide II of each fraction is eluted in about the same position. Since the results in Table 3 Show that these two polysaccharides are different, it is clear that in order to obtain pure 220 sodium chloride-soluble polysaccharide II, the 1.0 M-sodium chloride fraction step is essential. The 220 and 70° sodium chloride-insoluble fractions were resolved into four and three components, reSpectively. 51 by fractionation with sodium chloride. In both cases, the recovery of material was virtually quantitative in contrast with the recoveries from the DEAR-Sephadex columns. The polysaccharides of both insoluble fractions were precipi- tated at similar and definite sodium chloride molarities. The three polysaccharides of the 700 insoluble fraction have prOperties similar to polysaccharides B, C and D of the 22° insoluble fraction, again showing that the material extracted at each temperature is very similar. These eXperiments and those discussed above Show that sodium chloride can be used successfully to fractionate the apio- galacturonans. They confirm that the two insoluble frac- tions are mixtures of polysaccharides. Fractionation of pectic substances from plant material with inorganic univalent cations has been previously used successfully by Bhattacharjee and Timell (1965) and Zitko and Bishop (1965). The results in Table 4 show that there is a direct relationship between the D-apiose content of a polysacchar- ide and its solubility in sodium chloride solutions; the higher the D-apiose content, the greater the solubility. The apiogalacturonans which contained the greatest percen- tage of D-apiose were soluble even in 2 M-sodium chloride. However, they were readily precipitated by low concentra- tions of the bivalent calcium ion. In view of the very low D-galacturonic acid methyl ester content of all of the apio- galacturonans isolated, the sodium chloride fractionations 52 reported here are probably based on the ability of D-apiose to interfere with the formation of ionic bonds between the negative charges of the apiogalacturonan and the positive sodium ions. If precipitation occurred by the formation of ionic bonds between polysaccharide molecules via the sodium and chloride ions and if the D-apiose sterically shielded the negative charges on the polysaccharide molecules, those polysaccharides of highest D-apiose content would have the greatest difficulty to form such bonds and thus to aggre- gate. Consequently, these polysaccharides would be the most soluble in salt solutions. From Figure 3 it can be seen that hydrolysis of the 220 sodium chloride-soluble polysaccharide II with dilute acid resulted in the removal of the D-apiose from the poly- saccharide. The remainder of the molecule appeared to be a galacturonan as evidenced by its quantitative conversion to D-galacturonic acid by pectinase treatment. The galacturonan was relatively undegraded by the acid hydrolysis treatment as evidenced by its elution as a single peak from DEAE- Sephadex. However, only 30% of the eXpected uronic acid- positive material was eluted from the DEAR-Sephadex column. The pectinase hydrolysis results depicted in Figure 4 suggest that in the sodium chloride-soluble polysaccharides, the D-apiose is attached to the galacturonan throughout its length since these polysaccharides are resistant to pectinase- catalyzed hydrolysis. This is substantiated by the results 53 in Figure 5 which Show that these polysaccharides are readily hydrolyzed by treatment with pectinase if their D-apiose is removed. Since there is very little methyl esterification, the inhibition must have been due to the D-apiose Side-chains. If the D-apiose was concentrated at a few Specific points in these polysaccharides, a large pr0portion of the D-galacturonic acid should have been released by pectinase treatment before acid hydroly- sis. Since this was not the case, such a structure can be excluded. In the case of the sodium chloride-insoluble polysaccharides, their relatively small D-apiose content dictates that large portions of the galacturonan backbone must be free of D-apiose side-chains, thus making them susceptible to pectinase. In these polysaccharides, two types of structures are possible. Either the D-apiose is attached throughout the length of the galacturonan with sufficient Space in between the D-apiose molecules to allow the pectinase to act or all the D-apiose molecules are concentrated in one portion of the galacturonan leav- ing the remainder susceptible to pectinase. The available data do not allow us to make a choice. Because the apio- galacturonans from‘L.‘angg were degraded by the pectinase, we have assumed that they consist of a-(iw4)-linked D- galacturonic acid residues. The two apiogalacturonans isolated by Beck (1967) were both extensively degraded by pectinase. 54 The ability of D-apiose to confer resistance to pectinase on apiogalacturonans of high D-apiose content has not been observed before and could be physiologically significant. Recent studies have shown that an early consequence of pathogen infection in plants is the degra- dation of cell wall polysaccharides (Bateman, Van Etten, English, Nevins and Albersheim, 1969; English and Alber- sheim, 1969). Thus, one function of D-apiose 1N.£p gang; might be to protect the pectic substances from degradation by infecting pathogens. If D-apiose functions in this manner in'L.‘ngg£, it probably functions similarly in other plants that contain Deapiose. It will be of interest to survey such plants for apiogalacturonans. Pertinent to this is the isolation, by Ovodova gg‘gl. (1968), of a D- apiose-containing polysaccharide from three Species of the Zosteraceae family which also contained D-galacturonic acid. D-Apiose-containing polysaccharides have only been isolated from L.‘nggg and the three Species of the Zosteraceae family. In an early study on D-apiose, Bell 32,2L. (1954) suggested that the resistance of P. australis and L. marina fibers to natural decomposition may be due to the presence of a D-apiose derivative. The acid and enzymatic hydrolysis data indicate that the apiogalacturonans isolated consist of an unesterified galacturonan backbone to which are attached side-chains of D-apiose. Polysaccharides similar to those described here 55 have been isolated from plant material by ASpinall and Baillie (1963) and by Bouveng (1965). However, in these polysaccharides the side chains consist of sugars other than D-apiose. This type of polysaccharide where sugars are attached as side chains to a galacturonan backbone may be widely distributed in plants. Beck's acid hydrolysis and autohydrolysis data (1967) and his inability to detect disaccharides or higher homologues of D-apiose, led him to suggest that the D- apiose was attached to the galacturonans as monomeric side-chains. However, our formaldehyde data indicated that the D-apiose was partially substituted since only about 50% of the theoreticalquantity of formaldehyde was obtained on periodate oxidation. Only D-apiose will yield formaldehyde in these polysaccharides, and it would have to be substituted at position 3 or 3' to block formalde- hyde production during periodate oxidation. The theoretical amount of formaldehyde (i.e., 1 mole/mole D—apiose) was obtained if the apiogalacturonan was first hydrolyzed under conditions which resulted in the release of only free D- apiose. The most plausible eXplanation of the findings is to postulate the existence of acid-labile disaccharide side-chains of D-apiose attached to a galacturonan. Recently we have isolated a disaccharide of D-apiose from several apiogalacturonans by heating an aqueous 0.3% (w/v) solution of the apiogalacturonan at 1000 for 3 hr. at pH 4.5 (Part III). 56 The data in Table 1 Show that on a dry weight basis, 76% of the D-apiose of the cell wall of L. ngg; was not solubilized by our extraction procedure. Of interest will be the determination of the structure of D-apiose-containing polysaccharides remaining in the residue. If D-apiose is functioning to protect the pectic substances, the D-apiose remaining in the residue may be functioning similarly but protecting different polysac- charides, in which case different D-apiose-containing polysaccharides would be present. The isolation by Williams and Jones (1964) of a crude polysaccharide frac- tion containing D-xylose and D-apiose in the approximate ratio of 1:1 from the marine plant, L. marina, showed that other types of D-apiose-containing polysaccharides are present in nature. Alternatively, the D—apiose could be present in polysaccharides that are the same as those reported here but for unknown reasons are not extracted. A major obstacle to further extraction of the L. mlgg; cell wall is in developing sufficiently mild procedures for solubilizing the D-apiose-containing polysaccharides still in the residue. One possible approach is to use a purified enzyme to selectively degrade one type of poly- saccharide (e.g., cellulose) and determine if the remain- ing polysaccharides are now more readily extractable. PART 3 ISOLATION AND CHARACTERIZATION OF APIBIOSE FROM APIOGALACTURONANS 57 INTRODUCTION The occurrance of D-apiose (3-C-hydroxymethyl- ealdehydo-D-glycero-tetrose) as a component of several dif- ISerent types of compounds, isolated from a large number of p>lants, was briefly reviewed in.Part II. In the same earlucle, we reported the isolation of a series of apio- galacturonans from L. 3121133. The evidence presented indi- <3eated that the D-apiose was glycosidically linked as side- <311ains on galacturonans. Contrary to the conclusion of Beck (1967), the evidence also indicated that the side- cluains were disaccharide units of D-apiose rather than imxxnosaccharide units. Periodate oxidation of the intact aIpiogalacturonans indicated that the glycosidic linkage between the two D-apiose molecules of a sidechain was either 1->3 or 1—>3'. Further investigations on the structure of the apio- Esalacturonans from L. nggg, has led to the isolation of a (lisaccharide of D-apiose. We give the common name apibiose 'to this disaccharide. Apibiose is released from the apio- galacturonans by a very mild reaction which apparently is a hydrolysis. We report here the structural determination of apibiose and several parameters of the reaction involved in its release from the apiogalacturonans. 58 59 MATERIALS AND METHODS Materials Apiin and D-apiose were obtained as described in Plart II. Barium[:1h0] carbonate was obtained from New Ehngland Nuclear Corp. and was converted to sodium [180] t>icarbonate (5 uCi/umole) before use. Fungal pectinase was purchased from Sigma Chemical Company. The enzyme was {pixrified 30-fold over the crude preparation by the manu- tfsacturer who stated it still contained several other ellZymes. The Specific activity of this batch (Lot 125B- 0:350) as measured by Sigmna was 0.7 units per mg. of solid. ‘A ‘unit was defined as that amount of enzyme which liberated 1 lJmole of D—galacturonic acid per min. at 250 and at pH 4.0 with de-esterified citrus pectin as the substrate. ELLant Material ‘L. ELLE; was grown on modified medium V of Norris, Iqorris and Calvin, 1954, as described by Kindel (manuscript in.preparation). Large quantities °f.£-.E$22£ were obtained from the Battle Creek River at Bellvue, Michigan. The plant material obtained from the river was washed extensively and either used immediately or stored at -200. General Methods D-Apiitol was prepared from D-apiose by catalytic hydrogenation (Neal and Kindel, submitted to J. Bacteriol.). D-Apiose phenylosotriazole was prepared by the method of 60 Ifindel (1969) and D-apiose a-benzyl-a-phenylhydrazone was pxrepared as described by Schmidt (1930). Solutions were concentrated under reduced pressure by rotatory evaporation art temperatures less than 35°. Melting points were deter- unined with a Kofler micro hot stage (A. H. Thomas Co.) and aJre uncorrected. Optical rotations were determined with a Zeaiss Photoelectric Precision.Polarimeter 0.005O (Carl Zeeiss, Oberkochen, Wuerttemberg, Germany) at 22° and at .5778 mu and 546 m1. Molecular rotations were calculated from Specific rotations by the following formula: ; [a] x M.W. [M] = 100 Irlfrared Spectra were obtained with a Perkin~Elmer grating irlfrared Spectrophotometer, model 337, with air as the rEBference. Samples were prepared as potassium bromide ‘Pfisllets or as smears on potassium bromide pellets. Proton 11Magnetic resonance Spectra were obtained at 60 kHz and 100 1“Hz, with Varian.A6O and HA100 Spectrometers, reSpectively. ESamples were dissolved in deuterium oxide and exchangeable 'hydrogens were removed by concentration of the sample Several times from deuterium oxide. The final concentra- tion of the sample for Spectral analysis was 10% (w/v). The Spectra were obtained at ambient temperatures with tetramethylsilane as the external standard (T = 10). hass Spectra were obtained by the direct probe method with a LKB mass Spectrometer, model 9000 (LKB Instruments, Inc., 61 Stockholm, Sweden). Spectra were recorded at 70 ev., with an accelerating voltage of 3.5 kv., an ion source tempera- ture of 210°, and a filament current of 60 Ha. Radioactiv- ity was detected on chromatograms with a Packard strip counter, model 7201. All other radioactivity measurements were made with a Packard Tri-Carb liquid scintillation counter, model 3310, employing one of the following scin- tillation solutions: A) Bray (1960), or B) 2,5-bis-[2(5_ tert-butylbenzoxazolylU—thiOphene-toluene (4 g./l.). Chromatography Descending paper chromatography was used and was usually carried out with Whatman No. 3MM paper. This paper was prewashed with 0.1 M citric acid followed by distilled water unless otherwise noted. Unwashed Whatman No. 1 paper was used for methylated sugars. The following solvents were employed: A) ethyl acetate-acetic acid-formic acid— water (18:3:1:4, by vol.), B) propan-Z-ol-water (9:1. by vol.), C) propan-l-ol-ethyl acetate-water (7:1:2, by vol.), D) pyridine-ethyl acetate-acetic acid-water (5:5:1:3, by vol.), E) butan-i-ol-pyridine-benzene-water (5:3:lz3, by vol., upper phase), F) water saturated 2-butanone (redis— tilled), G) butan-i-ol-ethanol-water (10:3:3, by vol., upper phase). Sugars were detected on chromatograms with aniline hydrogen phthatlate (Partridge, 1949) or by the silver nitrate dip method (Trevelyan, Procter, and Harrison, 1950). 62 Gas-liquid chromatography was carried out with a F and M Corp. gas chromatograph, model 402, equipped with a hydrogen flame detector. The 6 ft. x 1/8 in. i.d. U- shaped borosilicate glass column was packed with acid washed and dimethyldichlorosilane treated Chromasorb W coated with 3% OV-i (Applied Science Laboratories, State College, Pa.) and was used at 110°. The internal standard was 2,3,4,6— tetra-O-methyl-D-glucose. Radioactive L. minor Radioactive L. minor were obtained by eXposing a small number of fronds in a closed chamber to increasing amounts of [luc] carbon dioxide over a 30 day period. The plants were grown under continuous incandescent light (approx. 100 ft-candles) at 22.240 on modified medium V. In the 30 day period, the number of fronds doubled every 3 dayso The radioactive carbon dioxide was administered every fourth day in amounts which varied from 5 uCi. at the beginning of the eXperiment to 100 001. at the end. The chamber was kept closed for 24 hr. after each adminis- [luC] carbon dioxide and was then Opened to the tration of atmOSphere for the next 48 hr. The total radioactive carbon dioxide administered was 415 uCi. The labelled plant material was diluted 5-fold with unlabelled L. E1225: grown under similar conditions, before use. 63 Isolation of Apiogalacturonans Apiogalacturonans were isolated as the sodium salts as described in.Part II. [13C] Apiogalacturonans were isolated from radioactive L.'nggg by the same procedure and had a Specific activity which varied from 14000 disin- tegrations min."1 mg.'1 apiogalacturonan to 1800 disintegra— tions min."1 mg.‘1 apiogalacturonan. Degradation of [14C] Apiogalacturonans [14C] Apiogalacturonans were degraded in screw—tap test tubes (13 mm. o.d. x 100 mm.) as described in the individual eXperiments reported. The solutions were then Spotted directly on unwashed Whatman No. 3MM paper and developed in solvent A. The dried chromatograms were scanned for radioactivity and the labelled areas were cut out and counted directly in scintillation solution B at 60% efficiency. The chromatograms all contained only the three labelled areas shown in Figure 1a. Greater than 90% of the starting radioactivity could be accounted for in these labelled areas. Isolation of Apibiose Either radioactive or non-radioactive sodium apio- galacturonates were dissolved in water to yield a 1% (w/v) solution. The pH of the solution was 4.3—4.5. The solu- tion was heated for 3 hr. at 100°, cooled and concentrated to approximately a 3% (w/v) solution. Three volumes of 64 acetone was poured Slowly into the stirred solution. The resulting suSpension was placed at 4° for 4 hr. and then centrifuged at 15000 x g. for 20 min. The supernatant solution was decanted and the precipitate was resuSpended in 1 volume of acetone. The precipitate was removed by recentrifugation at 15000 x g. for 20 min. The combined supernatant solutions were concentrated to a small volume and an aliquot was chromatographed in solvent A. The precipitate was dissolved in water to yield a 1% (w/v) solution and the above described treatment was repeated until there was only a small amount of apibiose present in the supernatant fraction. For the radioactive apio- galacturonans a single heat treatment was sufficient. However, in the larger scale non-radioactive isolations, 3-6 heat treatments were carried out. Radioactive apibiose was purified by preparative paper chromatography. The supernatant solution from the above described process was streaked on Whatman No. 3MM paper and the chromatograms were developed in solvent A. The driedchromatograms were scanned for radioactivity and the apibiose area was cut out and eluted with water. The eluate was concentrated to a small volume. The yield of radioactive apibiose was determined by counting an aliquot of the radioactive solution in scintillation solu- tion.A. The non-radioactive apibiose was purified by parti- tion column chromatography on powdered cellulose. The 65 combined supernatant solutions from the above described process were concentrated to a small volume. Whatman powdered cellulose (W. and R. Balston, Ltd., England, standard grade) was added to the solution until it became a thick suSpension. The suSpension was dried over phos- phorus pentoxide Lg ygggg.’ The cellulose column was pre- pared by a modification of the procedure described by Whistler and BeMiller (1962). The cellulose was suSpended in acetone by stirring and the fines were removed by decanting after standing for 5 min. The process was repeated twice. The cellulose was resuSpended in acetone by stirring and then poured into a glass column (5 cm. i.d. x 80 cm.) which contained a plug of glass wool on the bottom and acetone to a depth of approximately 20 cm. The column was completely filled with the slurry of cellulose and then the stopcock at the bottom of the column was Opened. The column was kept completely filled with the slurry until the desired amount was added. The column of cellulose (5 cm. i.d. x 40 cm.) was washed with 1—2 liters of acetone. The acetone was drained to the top of the column and then water saturated butan-i-ol was added. The column was equilibrated with 6 liters of water saturated butan-l-ol. The solvent was then drained to 1—2 cm. above the top of the cellulose and a circle of Whatman No. 5 paper was placed on tap of the cellulose. The sample was applied to the top in the dry state. A few m1. of water 66 saturated butan-l-ol was then added to facilitate removal of any trapped air. Another circle of Whatman No. 5 paper was placed on top of the sample. Solvent was added and a reservoir was attached. The column was then deve10ped at a rate of 1 m1./min. with 10 m1. fractions being collected. Aliquots of the fractions were Spotted on paper. The paper was developed by the silver nitrate dip method. The frac- tions containing the apibiose were pooled and concentrated to a syrup. The syrup was dissolved in water and treated with acid washed activated carbon (Darco G-60, Atlas Chem- ical Industries, Inc.). The charcoal was removed by fil- tration and the filtrate was passed through a Seitz filter. The filtrate was concentrated to a syrup and dried over phOSphoruS pentoxide Lg 13933. The yield of chromatograph- ically pure apibiose, [a]§%8-69.1° (05, water), calculated on the D-apiose content of the starting apiogalacturonans, was dependent on the number of times the degradation proce- dure was repeated. The cellulose column could be reused several times without alteration of the elution profile of the chromato- gram if the cellulose on which the sample was adsorbed was removed each time. Sodium Borohydride Reduction of [luC] Apibiose (peak 2) Radioactive apibiose was reduced with a 10-fold excess of sodium borohydride in water at 37° for 16 hr. 67 The solution was neutralized with acetic acid and then con- centrated to dryness. The borate was removed as the methyl- ester by distillation under reduced pressure. The reduced material was purified by paper chromatography on Whatman No. 3MM paper, first in solvent A and then in solvent D. The material migrated as a single peak in the latter solvent. Hydrolysis of ELTC] Apibiose and Reduced [1ch Apibiose (peak 2) Before radioactive apibiose and reduced apibiose were hydrolyzed, unlabelled D-apiose was added to the former and unlabelled D-apiose and D-apiitol were added to the latter. Both compounds were hydrolyzed with 0.1 N-sulfuric acid at 100° for 1 hr. The solutions were cooled and neutralized with sodium hydroxide to pH 6. The solutions were Spotted directly on Whatman No. 3MM paper and the chromatograms were deve10ped in solvent A. The chromatograms were scanned for radioactivity and the labelled areas were cut out and counted in scintillation solution B. Preparation of Apibiose PhenyLosotriazole Apibiose was dissolved in sufficient 5.0 M-sodium acetate buffer, pH 4.5, to yield a 2% (w/v) solution which was then heated to 80°. A 10-fold molar excess of phenylhydra- zine hydrochloride in an equal volume of the same buffer was heated to 80°. The solutions were mixed and heated for 1 hr. at 90-950. Three volumes of water at 22° was added to 68 the hot solution and the resulting precipitate was allowed to settle at 4° for 2-3 hr. The precipitate was collected by centrifugation at 35000 x g. for 20 min. The precipi- tate was dissolved in 1-3 ml. ethanol and 10 volumes of water was immediately added. After cooling to 4° for 4 hr., the resulting precipitate was collected by centrifugation and dried over phOSphorus pentoxide Ln 13233. The yield of amorphous apibiose phenylosazone, m.p. 91-93°, was 75-80%. (Found: 0.55.7; H,5.9; N,11.8; 022H27N407 requires 0.57.5: H.5-9: N,12.2%). The apibiose phenylosazone was suSpended in water to yield a 1.5% (w/v) suSpension which was then heated to reflux. A solution of 1.1 molar equivalents of cupric sul- fate pentahydrate in 0.5 volumes of water at 80° was added to the phenylosazone suSpension and the resulting reddish solution was refluxed for 1 hr. The solution was cooled to 4° and then filtered through Whatman No. 5 paper. The filtrate was concentrated to a small volume and then streaked on several sheets of unwashed Whatman No. 3MM paper. The papers were developed in solvent A. The fluorescing bands at RF 0.69, detected by 366 mu ultra- violet light, were cut out and eluted with water. The eluate was concentrated to 5-10 ml. and then continuously extracted with diethyl ether for 30-36 hr. The diethyl ether solution was treated with Darco G-60 and then fil- tered. The filtrate was concentrated with a stream of air 69 until the solution became cloudy. Crystallization occurred on standing at 22°. The resulting colorless needle-like crystals, m.p. 116.5-117.0° and [a]§$8-80.0° (05, water) were obtained in a yield of 25% from apibiose phenylosazone, for an overall yield of 20%. The apibiose phenylosotriazole was recrystallized from diethyl ether by solution in water and re-extraction with diethyl ether. (Found: 0.51.9; H,5.8; N,11.4; C16H21N307 requires 0.52.3; H,5.8; N,1i.5%). Periodate Oxidation and Determination of Formaldehyde Samples of apibiose and apibiose phenylosotraizole (4 to 17 mg.) were dissolved in 1 to 2 ml. of 0.05 M- sodium acetate buffer, pH 5.0, and then a 2.5-fold theoret- ical excess of sodium metaperiodate, in an equal volume of water, was added. The solutions were kept at 22° for 2 hr. in the dark. Preliminary eXperiments had Shown that for- maldehyde production was complete after 1 hr. The solutions were passed through a column (1 cm. i.d. x 7 cm.) of Dowex 1-X8 (200-400 mesh, acetate form). The formaldehyde quan- titatively passed through the column and was determined with chromotropic acid (recrystallized once from aqueous 50% (v/v) ethanol). Methylation.AnalySis The methylation procedure was essentially that of Hakamori (1964). However, the methylsulfinyl anion was 7O generated as described by Sandford and Conrad (1966). The methylated compounds were extracted from the reaction mix- ture with chloroform. The extent of methylation, as a func- tion of hydroxyl absorption, was determined by infrared Spectroscopy. Methylated compounds were hydrolyzed with 1 N-sulfuric acid for 1 hr. at 100°. The hydrolysates were neutralized with sodium hydroxide to pH 6 and then 9 volumes of ethanol were added. The insoluble sodium sul- fate was removed by centrifugation and the supernatant solutions were decanted. The supernatant solutions were concentrated for chromatography. Standard 2,3,3l-tri-O-methyl-D-apio-D-furanose (Halyalker, Jones and Perry, 1965) was isolated from the supernatant solution of a hydrolysate of 1 g. of methylated apiin by partition chromatography on a column (2 cm. i.d. x 41 cm.) of powdered Whatman cellulose. The column was poured and equilibrated with water saturated redistilled 2-butanone (Sandford and Conrad, 1966). The sample was prepared and applied to the column as described for the isolation of non-radioactive apibiose. The column was then developed with the same solvent. Fractions (3 ml./14 min.) were collected and aliquots were chromatographed on Whatman No. 1 paper in the same solvent. The sugars were visualized with aniline hydrogen phthalate. The fractions containing chromatographically pure 2,3,3'-tri-O-methyl-D- apio-D-furanose were pooled and concentrated to a small volume. 71 Standard 2,3,4,6-tetra-0-methyl-D-glucose was pre- pared from D-glucose by this procedure also. Isolation and Pectinase Hydrolysis of Partially DegradedLEFC] Apiogalacturonans The precipitate obtained an addition of acetone to the thermally degraded [1uC] apiogalacturonans (see above section on Isolation of Apibiose) was designated partially [14 degraded 0:]apiogalacturonans. This precipitate was dissolved in water, concentrated to a small volume and freeze-dried. One (1.0) mg. of partially degraded [140] apiogalac- turonans were hydrolyzed with 2 mg. of pectinase for 2 hr. by the procedure described in.Part 2. The hydrolysis was terminated by heating for 1 min. at 100°. The denatured protein was removed by centrifugation. The supernatant solution was Spotted directly on unwashed Whatman No. 3HM paper and deve10ped in solvent D. The chromatograms were dried, scanned for radioactivity and the labelled areas were cut out and counted in scintillation Solution B. Percent hydrolysis was the percent of the total recovered disinte- grations/min. in D-galacturonic acid. RESUDTS Degradation of‘EluC] Apiogalacturonans When sodium [14C] apiogalacturonates isolated from L. minor were heated in water at 100° and then chromatographed 72 in solvent A, two radioactive peaks of high RF were detected (Figure 1). In the unheated control all of the radioactivity remained at the origin of the chromatogram. The same two peaks were obtained from all four apiogalac- turonan fractions obtained by ammonium oxalate extraction (Table 1, Part 2). However, treatment of the 70° water fraction and the residue fraction in a similar manner did not yield these two compounds. Because of the high D- apiose content of the 22° sodium chloride soluble IIa (22SCS-IIa) apiogalacturonan (Table 3, Part 2), this poly- saccharide was used primarily for the characterization of the degradation reaction, where degradation is defined as the percent of the total re0overed disintegrationS/min. in peaks 1 and 2. This degradation reaction was dependent on the temperature and the length of the heating period (Figure 2). Degradation at 1000 occurred very rapidly and reached a final value of 40% degradation after 5 hr. There was still extensive degradation at 80°. However, the degrada- tion after 7 hr. at 60° was only 10% of that which occurred at 100°. The degradation reaction was also dependent on the pH of the solution (Figures 3 and 4). The rate of the degradation declined steadily from pH 3.5 to 6.0 until it dropped to essentially zero at pH 6.5. There was also a change in the ratio of the two compounds obtained during 3 7 .moonuo: 02m mamaaopma map SH popaaomop ma ompmmpp who: mammmm one .Anv omm um hams no Adv .as n how 000H pa panda: ohms m.: mm as A.HS\.wS my Hopes SH camoHSpoaHmonaa we SoapsHom a mo A.Ha a.ov mposwaa< .aozaa .A aoam opaSOMSpomHawoaam moaau asdpom pmpmamop maaaaaomp mo sapw0pmaoano momma a mo mmom .H oaswam 7b. me See :ch _ xdwm Sound N yawn 0 38324 2: LI... a - 26.8 2: 10103190 asuodseg 75 .N 0cm a mxmmm Ca .caa\mcodp imamopaamap poaoboooh Hmpop exp 00 amooaoa asp ma Soapapmamop paoonom .mUOSpoa 0cm maaaaopaa 05p Ca confinomoo mm papacy» who: mammmm one .oaap mean any ad popoaaaoo who: Ham was» and: a scam SH oopaapm opos- oam A‘V O00 0mm . AOV oow . ADV oooa pm .a: 5 0mm .m .m .H pom pupae: ones mammma one .mmmmm some now moms who: .Ha H.0 0o mposvaam .m.: was Soapsaom as» mo ma one .HE\.wB m mo Sofipmapzoo 1200 a pm Hopes ad 0o>aommae was .pamm Badman map as .moaamsoommmfloa use .mHSpmaoaSop 0am asap no mofipom50 a ma mHHlmomNm QmQOHSpomHmmodam mvdag Co aoapmpmawma .N oasmam 76 ’- J l 0 q- . (\l uogiopolbeg iUSOJSd 3 4 Time (hr) 77 .N van a madam ca .afia\mmoHpaamop:HmH0 pmaoboooa Hmpop amp 0o pmooaoa 0:» ma moapmpmamoo acooaom .mvonpoz 0mm mandamus: on» ma popdpomou mm panama» who: mammmm one .oaau mean can pm oopoaaaoo mama Ham was» has a Scam SH popampm one: 0am oooa pm acumen ohms madman one .mmmmm memo pom poms cams .Ha H.0 mo mposaaa< .Ha\.wa m 00 godpahpsoosoo a pm .3: 03m one .23 no .A! oo 43 Rm .3 mi .30 m8 ma .aotsp assessors Baammmpoala m0.0 ca nobaommap was ovaamzoommmaoa on» 00 wash Baacom one .ma 00 :oHpocSm H mm mHHumommm :m20p5pomamonam moaau do godpmpmawmu 00 mean 059 .m masufim 78 no Ia Q mo Ia 0.0 Ia he Ia Om Ia see as: O _ O N Om iuealed uogiopoibeo 79 .m oaswam ad Dopedaom dump amp 5090 pmpHHSOHmo who: Scapmpmammp mo mouse one .ma 00 soaposz ‘ a ma mHHlmummN masoaSpomHmonaa modag 00 godpmpmammp 0o mama age .3 oaswam 80 8.0 7.0 l L T l 4.0 6.0 pH 5.0 O, V N 0 (0 0| x ugw Jed uogwpwbao waxed 81 the degradation as the pH of the solution decreased (Table 1). As the pH of the solution was decreased from 5.5 to 3.5. there was an increase in the amount of peak 1 in rela- tion to peak 2 as well as an increase in the total percent degradation. The rate and limit of the degradation were slightly inhibited in the presence of salt (Figure 5). The limit of the degradation obtained in 0.2 M-potassium phOSphate buffer, pH n.5, was 90% of that obtained in water. The rate of the degradation was also a function of the concentration of the apiogalacturonan (Figures 6 and 7). The rate decreased as the concentration decreased from 10 mg./m1. to 1 mg./ml. The rate obtained at 1 mg./m1. was 67% of that obtained at 10 mg./ml. There was also an decrease in the ratio of peak 2 in relation to peak 1 as the concentration increased. After 90 min. at 100°, the ratio was 10.#, 8.2 and 6.5 at 1 mg./ml., 5 mg./m1. and 10 mg./ml., reSpectively. The degradation resulting with con- centrations below 1 mg./ml. could not be measured accurately and concentrations above 10 mg./m1. were prohibitive because of the quantity of material needed for such assays. Heating sodium [luC] apiogalacturonate ZZSCS-IIa in 0.5% ammonium oxalate, pH 6.2 (3 mg./ml.), for 6 hr. at 70° resulted in 3.75% degradation. 82 Table 1. The degradation products of [14C] apiogalacturonan ZZSCS-IIa as a function of pH. Aliquots (0.1 m1.) of the sodium apiogalacturonate were heated for 180 min. at 100° at pH 3.5, 4.5, 5.5. 6.0, and 6.5 and at a concentration of 3 mg./ml. The individual assays were treated as described in the Materials and Methods. Percents are eXpressed as the percent of the total recovered disintegrations/min. ' Peak 2 Total pH Peak 1 Peak 2 Peak 1 degradation (%) (%) (%) 3.5 10.5 27.8 2.64 38.3 4.5 6.3 30.9 4.91 37.2 5.5 3.1 26.4 8.52 29.5 6.0 1.7 8.b 4.9% 10.1 6.5 0.7 3.0 “.28 3.7 83 .m 82¢ a mema ca .caE\mnoHpmaw umpzdmao ooaoboomp Hmpop 0:» mo unooama on» ma sodpmomamoo unmopom .moonpoz one mamapopwz on» ma omnaaommp mm ompmoap mums mammmm one .maap meow on» no oopoaaaoo ohms Ham pomp mm: m zoom ca omppmpm who: one oooH um venom: mam: mammmm one .mmmmm comm you now: mam: .HE a.o mo wposwafi< .Ha\.wa m we sod» unannoocoo a pm 82m. m3 ma pm Ham .opmgamosa asammmpoa A‘v :2 N6 paw . “CW 1. H.o .AOV .Hopms 2H om>Hommac mos ogfimnoommmaoa on» ,Ho pdmm azaoom one .mHHnmommm cocoazpomamm noHQm modau mo zofiumomamoo m£p :o codpmapsmozoo pamm mo pomwmo mse .m onswam 84 Lo 4 ' 04 1 k 004 oo< cl) \ v 8 uou‘opmfiec] waxed 0 Time (hr) 85 .N was H mxmma ca .cfia\m:o«pwhwmp:amao omhoboooa Hmpop 0:» mo pcmohma on» ma aoapmowamoo pamopmm .moonpoz paw mamdampmz on» :« omnaaommo mm ompmoap ohms mammmm mss .maap mamm on» no Umpoaaaoo ohms Ham was» as: a £05m 2H oophmpm ohms dam oooa pm acumen who: mammmm one .HS\.wE AOV o.oH was .AQV o.m .AOV of" mo mcoapmapsmosoo pm .m.: mm as Hops: ca oobaommao was moaamnoommmaoa map no pamm asfiuom 0:9 .soflpmapCmosoo mo Goapondm m mm mHHamomNN chOASpomHmwOan mvdau mo coapmomammo mo mama one .0 chowdm 86 ES .2: Om ow ON 0 _ _ _ o O 4 O O. O 4 :59: _ O 0 ON :59: m a .532 o. [w waxed uouopmbag 87 .0 oasmdm 2H ompoaamo muse on» song oopmHSOHmo cams sodomomammo no mode one .sodpmapcmocoo mo soapoQSQ a ma mHHumommm chOHSpomemoHam hoeaw mo soapmomawmo yo mama one .n maswam 88 l l 6 [Cone] mg/ml I i 4 l I <1- N 00 0| x ugw Jed uouopmbao wowed 89 Identification of Radioactive Peak 1 Peak 1 was chromatographed in solvents A, B, C, and I). In all four solvents, it migrated as a single peak with ‘tkm same RF as D-apiose. When chromatograms were treated wwith aniline hydrogen phthalate, peak 1 developed as a srellow Spot which fluoresced when viewed with ultraviolet ]_ight (max. 366 mu). Both of these results are character- j.stic of D-apiose. Therefore it was concluded that peak 1 vvas D-apiose. jgdentification of Radioactive Peak 2 Peak 2 chromatographed as a single peak in solvents A . B, c, and D. It had an RD_aplose of 0.53. 0.65, 0.75, Etnd 0.95 in solvents.A, B, C, and D, reSpectively. When czhmomatograms were treated with aniline hydrogen phthalate, tweak 2 developed as a yellow Spot which fluoresced when \riewed with ultraviolet light (max. 366 mu). On hydrolysis With 0.1 N-sulfuric acid, peak 2 was converted quantita- tively to a compound which was chromatographically identical tC> D-apiose. The crystalline a—benzyl-a-phenylhydrazone d-'533:'ivative of this compound had a melting point of 137-1380 2 1&6 01? o 22 D-apiose had a melting point of 137-138 and [“3546- ' 2 and a [0.35 -86.3° (02.7. DYridine). The same derivative 90.1° (cu, pyridine). The mixed melting point of these two derivatives was 137-1380. Acid hydrolysis of sodium boro- Ytydride reduced peak 2 resulted in the release of equimolar (luantities of D-apiose and D-apiitol. Similar hydrolysis 90 of the phenylosotriazole derivative of peak 2 resulted in the release of equimolar quantities of D—apiose and D- apiose phenylosotriazole. On the basis of these data, it was concluded that peak 2 was a disaccharide of D-apiose (apibiose). Chemical Characterization of Apibiose Both apibiose and apibiose phenylosotriazole were oxidized with sodium metaperiodate and the amount of for- maldehyde released was determined. The results of these eXperiments may be seen in Table 2. The free disaccharide was oxidized with the release of 1 mole of formaldehyde per mole of disaccharide. Periodate oxidation of apibiose phenylosotriazole resulted in the release of 2 moles of formaldehyde per mole of derivative. Methylation analysis of apibiose and apibiose phen- ylosotriazole resulted in the isolation of a single methyl- ated sugar from the latter and two methylated sugars from the former. The fast migrating component from methylated apibiose and the methylated sugar component from methylated apibiose phenylosotriazole were indistinguishable from 2,3,3'-tri-O-methyl-D-apio—D-furanose as determined by paper and gas-liquid chromatography (Table 3). These methylated sugars also co-chromatographed with 2,3,3'-tri- O-methyl-D-apio-D-furanose as a single undistorted peak in the gas-liquid system. 91 Table 2. Periodate oxidation of apibiose and apibiose phenyl- osotriazole and determination of formaldehyde. Periodate oxidations and determination of formaldehyde were performed as described in the Materials and Methods. umoles umoles moles H2C0 Compound Compound HZCO mole of compound Apibiose EXpt 1 62.41 69.56 1.11 EXpt 2 38.30 39.73 1.04 Apibiose Phenylosotriazole EXpt 1 13.83 28.20 2.04 EXpt 2 11.17 22.97 2.06 EXpt 3 10.90 22.13 2.03 EXpt 4 17.70 35.92 2.02 92 Table 3. Chromatography of methylated D-apiose from apibiose, apibiose phenylosotriazole, and apiin. The methylated sugars were prepared as described in the Raterials and Methods. Solvent Apibiose Apibiose system Apiina phenylosotriazole 1 2 Paper Chromatography A 0.95 0.96 0.96 0.83 E 0.96 0.94 0.94 0.85 F 0.97 0.97 0.97 0.74 G 0.93 0.92 0.93 0.82 Gas-Liquid Chromatography 0.32 0.32 0.32 -- Note: Paper chromatographic mobilities are quoted relative to 2,3,4,6-tetra-O-methyl-D-glucose. are quoted relative to the same standard. a2,3,3'-tri-0—methyl-D-apio-D-furanose. Retention times 93 .mmoHamua Cd .zaa\m:oapmamopcfimao coaoboooa Hmpop ozp no psooaoa oz» ma mam imaoapzx pcooaom .moogpoz cam mamaaopm: on» ca vmnanommp mm Umpmoap ohms mammmm one .oafip memo map pm popmaaeoo mam: Ham pmSp mm: m £05m CH oopamum who: mammmm one .oooH pm ompmmz mam: :oHpsHom .Ha\.ws H m 80 A.Ha m.ov mposwaa< .23 m5 mam .AS m.m .on m.m ma pm mumsamonu anammmpoanm mo.o :« omOanHQm modau no mammaoaomm .w opsmfim 94 45 I I in. IQ“). O -N POW—8 :5 £35 ‘ 9 do H O —- —9 — mg 0 (3 H H o 01 _. \ “0 l0 0 0 J l \l 0 LO 00 m S!SK|OJpKH weaned Time (min) 95 Hydrolysis of Radioactive Apibiose Radioactive apibiose was hydrolyzed with 0.05 h- potassium phoSphate at pH 2.5, 3.5, and 4.5. AS the results in Figure 8 indicate, after 190 min. at 1000, there was very little hydrolysis at pH 4.5 and 3.5 (2.4 and 3.7%, reSpectively) while at pH 2.5 there was 42; hydrolysis. Physical Characterization of gpibiose The infrared Spectrum of apibiose was identical to that obtained for D-apiose except for an absorption band at 825 cm'1 in the apibiose Spectrum (Figure 9). The infrared Spectra of the phenylosotriazole derivatives of these two compounds (Figure 10) were more complex than the free sugars. The Spectrum of apibiose phenylosotria— zole revealed bands at 825 cm"1 and 1278 cm’1 which were absent from the D-apiose phenylosotriazole Spectrum. The Spectrum of the D-apiose phenylosotriazole revealed an intense band at 1232 cm’1 which was absent from the Spec— trum of apibiose phenylosotriazole. The Spectra between 4000 cm"1 and 1400 cm‘1 of all of these compounds was non- informative and contained no distinguishing bands. The proton magnetic resonance Spectrum of apibiose phenylosotriazole may be seen in Figure 11. There was no detectable resonance between 0.0-1.3 T and 6.3-10.0 T. The Spectrum contained two distinct regions. Integration of the 1.5—2.3 T region indicated that this region contained 96 wow .maampoo ampsmsaamaxm pom muospo: one mamaaopmz .A3\3V RH was mpoflaoa ooHSOMD azammmpoa on» :« oaaamm mo canoes one .mmOaDan 6cm omOammim no anypomam nonmath .m oasmdm 00¢ 9-68 35:68... 97 u-d omofioad 6,854-0 000 00h 000 000 000 _ 00. _ 00w. 00m. — u d _ d K) 100.. 1 Ch. 00. 0m. 0m. 0.. eouoqiosqv 96 mom .maampoo ampsmsaaoaxo pom moonpoz one mamaaopmz .A3\3V RH mm: mpoaaoa moasoan azammmpoa on» ad maaamm mo panoam one .mmoHnaam one omOanim mo asapooam omamamnH .m oasmam 9-88 3:638“. 8.. 8n ooo ooh oom com ooo. oo: oom. 8n. 4 a _ _ . _ q a 8 l 1 0». $0523. om. } L e on. v. 7 9 6854-0 0». om. (‘ 2 i 0.0 eouoqiosqv 98 .Hampoo ampsoaaaoaxm you moonpoz mam mamaaopmz mom .A3\3V mm.o mm: mpoaaoa ocaaoan anammmpoa on» ad oaasmm mo pssosm one .Alllv odoum«HuOmoazCo£Q omofindam tam Aiilv oaoumdapomoahcona moodamlo mo aanpooam nonmamsH .oa mammam 99 o‘---—--------- ‘-~.---------------- \ \ ‘- \- N '1- ”—--’---- ’l ” 2, a---‘ ---- -----‘ ’-——"’-- " 5 ~~--~---- '--‘---' ‘-—------—--- ‘ e~ - .- (-’---------- i *- ~-~- -----------—---- “""'" -=..- ----- ~------ -u--- ¢=::.- " v"-‘- S ------~ -‘ ---------' " r‘"""""":'n r I a ------- ---¢--"- 0.0 r- O O O N If) V’ .IO- eouoqiosqv -~ - " - .I - ._‘_-_]=-a. I 2 l l O O O '0 ‘9. N LOOh IZOO I |00 IOOO 900 700 600 500 400 Frequency (cm") I300 100 .Nmz ooa pm posfimpno was asepooam one .A>\z .Roav ovaxo asfiampSoo SH odommappomOHmcmzu omOaDHmm mo sapwooam mosmcomoa oauosmma Sopoam .HH opzmam 101 Cs Sam 5 he ho ho 11W ( 4 1(4144141 441 44414 44444<<44 J11 «1 41 {1 4 1 A <144444H44 {(#44 ‘41 4 J a a a a a a x i i _ . a 4 a a _ . I I I ow I I _ J0. Jn NI J: ‘II' 08. . _ a L s a P F F a _ --L----r-, My -r--r»~.-:e+- fit--. Lari).-- H, r .----H.rr-s . H r ,1- it... n46 - _ n6 m... n.» E; Edd 102 Six protons. There was a one proton singlet at 1.59 T, a two proton multiple at 1.63-1.78 T, and a three proton multiple at 1.82-2.20 T. The other region at 4.3-6.3 T contained the remaining ten protons. This region contained a one proton doublet (J = 2.6 Hz) at 4.50 T, a large HOD peak at 4.87 T, a partially resolved area at 5.3-5.8 T, and a two proton Singlet at 6.11 T. EXpanSion of the par- tially resolved area (5.3-5.8 T) of the Spectrum led to the increased resolution seen in Figure 12. This area of the Spectrum contained a two proton singlet at 5.78 T, a two proton Singlet at 5.67 T, a one proton doublet (J = 2.6 Hz) at 5.64 T, and a two proton.AB quartet (J =rv10.0 HZ) at 5.62 T. The results are summarized in Table 4. Mass Spectral analysis of apibiose phenylosotria- zole did not yield a molecular ion under the conditions employed for ionization. However, there was an intense peak at m/e 336 which correSponds to E-31. Under the same conditions, a molecular ion was obtained for D-apiose phenylosotriazole. The mass Spectrum obtained with apibi- ose phenylosotriazole recovered from deuterium oxide was identical to that obtained with material which was never in deuterium oxide. This result indicated that there was no permanent exchange of C-H hydrogens during the recording of the proton magnetic resonance Spectrum. 103 .Nm: ooH pm pocamppo mmz asapoomm one .e mm.m-mm.m sooaaoa mHommHapomoamcosa omoHDHQm no asapooam mosmgomoa capocmms Sopoam .Na oaamam 104 DQM mad :3 .210— - nod ‘ owe 9v.“ a: TI! 00.0 n. .0 mm m , and CL San. 00.0 09¢ Table 4. 105 Summary of the proton magnetic resonance data obtained at 100 MHZ for apibiose phenylosotriazole in deuterium oxide. Peak assignments are numbered according to the diagram below. Position Number of Coupling (T) protons Type constant Assign. (HZ) 1.59 1 singlet —— H-11 1.63-2.20 5 multiple —- H-12,13 14,15,16 4.50 1 doublet 2.6 H-1 5.62 2 A8 quartet 10.0 ___a 5.64 1 doublet 2.6 H-2 5.67 2 singlet -- ---a 5.78 2 Singlet -- ---a 6.11 2 singlet -- ___a 81These signals are from protons 3,4; 5,6; 7,8; or 9,10. ti. 6 (TV ®HC—:—0D l H 0 o H 3‘69 6 /© \\ (96 H C? I) ®H HC-OD H . CD F" I CD on 106 Characterization of Partially Degraded [luC] Apiogalacturonans When partially degraded [luC] apiogalacturonans were chromatographed in solvent A, all of the radioactive material remained at the origin. Pectinase hydrolysis of partially degraded [luC] apiogalacturonans resulted in a 75% conver- sion to D-galacturonic acid. Further characterization was not attempted. DISCUSSION The apiogalacturonan (22 SCS-IIa) used in most of this work is a representative member of the group of apiogalac— turonans isolated from L. mig93_(Part II). All had a low content of esterified D-galacturonic acid residues. The low ester content and the pH profile depicted in Figure 4 for the thermal degradation reaction of this apiogalacturonan indicate that the degradation reaction reported here is a hydrolysis reaction rather than an elimination reaction such as that described for pectins by Albersheim (1959). The failure to detect any unsaturated degradation products and the ready conversion of the galacturonan residue to D-galac— turonic acid by pectinase treatment also Speak against an elimination reaction. However it does not appear to be a Simple acid hydrolysis reaction. The relatively mild acid conditions needed for degradation suggest that the reaction may be an intramolecular hydrolysis involving the free 107 carboxyl groups of the D-galacturonic acid residues. The stability of apibiose to hydrolysis at pH 3.5 and 4.5, conditions which result in the rapid release of this com- pound from the apiogalacturonans, supports this suggestion. However, the rate of the degradation and the ratio of the degradation products were also a function of the apiogalac- turonan concentration, results which indicate that there may be some intermolecular hydrolysis. The disaccharide of D—apiose was the main degradation product isolated, Whether the D-apiose in the apiogalac- turonans exists entirely as disaccharide sidechains which are then partially hydrolyzed to D—apiose during and/or after hydrolysis from the apiogalacturonans, cannot be determined from the data. The periodate oxidation data reported in Table 7, Part II, also indicate that the D-apiose is almost entirely in the disaccharide form. The limit of the percent degradation for all of the apiogalacturonan fractions approached the percent D-apiose of these fractions, indicat- ing that whatever the mechanism, the D-apiose appears to be attached to the galacturonan backbone in a Similar manner in all of the fractions. All of the above described data lead to the conclu- sion that there is an unusual structural feature of these apiogalacturonans which causes the apibiose:galacturonan glycosidic linkage to be extremely susceptible to hydrolysis. The apibiose can only be linked to the D-galacturonic acid 108 residues at position 2 and/or 3 since the galacturonan is linked a-(1-94). If the apibiose was attached to the hydrOXyl group at C-3 and the D-galacturonic acid residues were in the conformation indicated (I), it would then be in close proximity to the carbOXyl group. However, this structure and conformation may not be required for hydroly- sis Since there may be deformation of the apiogalacturonan due to the heating which could result in conditions favor- able to hydrolysis when the apibiose is attached to C-2 of the D-galacturonic acid residues. It would be of interest to determine if the galacturonans isolated by Bouveng (1965), ASpinall and Baillie (1963) and ASpinall and Fanshawe (1961), which also contain a large percentage of neutral sugars as side-chains, also undergo a degradation of the type reported here. The thermal degradation of apiogalacturonan 223C8- IIa in 0.5% (w/v) ammonium oxalate, pH 6.2, was very Slight, being 3.75% after 6 hr. at 700. In Part II, the possibility of the partial degradation of the 700 ammonium oxalate fractions due to the elevated temperatures was noted. This result indicates that any degradation of the type described in this report was probably minor. The failure to detect apibiose or D-apiose when the residue fraction was heated does not imply that the remain- ing 76% (Table 1, Part II) of the D-apiose of the cell wall is in a different form. First, the degradation may not 109 occur in the solid state where the carboxyl groups may be unavailable due to complexing with cations. Secondly, if the carboxyl groups of uronans have an essential role in the degradation of the apiogalacturonans and the D-apiose remaining in the cell wall is a component of polysaccharides which contain only neutral sugars then these polysaccharides would not undergo a degradation such as described here. Therefore nothing may be stated at this time as to the state of the D-apiose remaining in the residue fraction. Apibiose was oxidized by sodium metaperiodate with the release of 1 mole of formaldehyde per mole of disacchar- ide. This result and the periodate results obtained earlier with intact apiogalacturonans (Table 5, Part 2), indicate that the glycosidic linkage between the two D-apiose mole- cules is either 1—93 or L—93'. It is of interest to note that this result with apibiose also indicates that the intermediate formyl ester (IV) was essentially stable under the conditions of the oxidation. The data obtained from the periodate oxidation of apibiose phenylosotriazole permitted a choice to be made between the two linkage alternatives. Periodate oxidation of (II) should result in the release of one equivalent of formaldehyde while the oxidation of (III) Should result in the release of two equivalents of formalde- hyde. The data in Table 2 show that two equivalents of formaldehyde were released. Therefore the glycosidic link- age between the two D-apiose molecules is i_+3' and 110 HO\C¢O H H OH o_.__ O _.__o H H H OH (I) H\ C=N c=N/ __ HOHzC—C-CHZOH HZf—C-OH H 0 o H 0 o cnzon H CHZOH H H H CHZOH H H OH OH OH OH (II) (111) H O HOH C 4\ . H CH2 H O Ofi I I C o E I H \ OH OH IO“ 0 PH 5.0 H 332° f OH OH (V) OH H (Iv) 111 structure (III) is the correct one for apibiose phenyloso- triazole. The methylation data indicate that the non-reducing terminal D-apiose of apibiose has the D-apio-D-furanose configuration. This is the same configuration as that obtained by Halyalker e_t__a_l. (1965) for the D—apiose in apiin, the only other naturally occurring compound for which the configuration at C-3 of D-apiose has been determined. The stereochemistry of the reducing terminal D—apiose at C-3 was not established. However,since apparently the natural isomer of D-apiose is the D,D isomer, the reducing terminal D-apiose may also have the D-apio-D-furanose con- figuration. The slower migrating component from the hydrolysates of methylated apibiose is from the reducing end of the disaccharide. The methylated compound is 2,3- di-O-methyl-D-apiose. Molecular rotations have been used to indicate the stereochemistry of anomeric linkages. Halyalker gt a}. (1965) extrapolated the theory of Klyne (1950) to the case of apiin and concluded that the linkage of the D—apiose to the 7-O-D-glucosyl-apigenin had the 3 configuration. Klyne's theory states that the contribution of a carbohyd- rate moiety to the total molecular rotation of a compound is approximately equal to the molecular rotation of the methyl glycoside of the carbohydrate moiety. That is A rotation .. En] R-carbohydrate - [In] R [M] methyl glycoside of carbohydrate 112 For sugars of the D-series, the d-methyl glycosides have a more positive rotation than the B-methyl glycosides. In fact, the d-methyl glycosides of D-sugars usually have a positive rotation or a rotation close to zero while 3- methyl glycosides have rotations of -1000 to -300°. The molecular rotations of identified methyl glycosides of D- apiose have not been determined. Williams and Jones (1964) isolated a chromatographically pure methyl glycoside of D-apiose which had a [QJgu-IOIO ([M]%4-165.6o). However they did not characterize the configuration at C-1 or C-3. Those isoprOpylidene derivatives of D-apiose which have the d configuration all have a positive rotation (Carey, Ball and Long, 1966, and Ball, Carey, Klundt and Long, 1969). The theory of Klyne (1950) is not directly applic- able to the determination of the anomeric configuration of D-apibiose phenylosotriazole since the attachment of the second D-apiose molecule to D-apiose phenylosotriazole creates a new asymmetric center at C-3 of the D-apiose phenylosotriazole component. The observed molecular rota- tion for apibiose phenylosotriazole, -294°,is due to the second D-apiose molecule plus the component due to the asymmetric center at C-3 of the D-apiose phenylosotriazole. Since its rotation is zero, D-apiose phenylosotriazole cannot be used to assess the rotation contribution of the assymmetric center at C-3. D-Erythrose phenylosotriaZole 113 could be used, however this compound is not known. The case of the free apibiose is different from that of apibiose phenylosotriazole. Since there was no detectable mutarotation, the configuration at the reducing end of the molecule must have attained equilibrium before the compound was concentrated to a syrup. The molecular rotation of equi- librated D-apiose is only slightly positive (less than 10°) fichmidt, 1930). Therefore, keeping in mind that the reducing terminal D-apiose molecule can only equilibrate between three forms whereas free D-apiose can equilibrate between five forms, one may still conclude that the major component of the observed 22 578 to the non-reducing D-apiose molecule. This means that this rotation of the disaccharide, [M] -194.9°, is probably due molecule is contributing a negative rotation. In view of the positive rotations for the isopro- pylidene derivatives of D-apiose, which have the c configura- tion and the fact that methyl a-D-erythrofuranoside and B-D— erythrofuranoside have molecular rotations of approximately +199O and -217°, reSpectively (Halyalkar, Jones and Perry, 1965), the negative rotations obtained for apibiose and api- biose phenylosotriazole indicate that the anomeric linkage has the 8 configuration since it is doubtful that the d-methyl glycoside of D-apio-D-furanose would have such a large nega- tive rotation. Assignment of protons to all of the signals in the proton magnetic resonance Spectrum of apibiose phenyloso- triazole cannot be made since the Spectra of a sufficient number of model compounds have not been determined. The 114 assignments which have been made were based on literature precedents for proton magnetic resonance Spectra of organic compounds in general (Silverstein and Bassler, 1964, and Morrison and Boyd, 1966, and references therein) and of carbohydrates Specifically (Hall, 1964, and references therein). From the proton magnetic resonance Spectrum the apparent coupling constant between the H-1 and H-2 protons of the non-reducing D-apiose molecule was calculated to be 2.6 Hz. Assuming the modified constants of Abraham.g£_gl. (in Hall, 1964) for the Karplus equation (Karplus, 1959) are valid for this system, values of 56t5° (00305900) and 121t5° (90°$¢$180°) were calculated for the dihedral angle between H-1 and H-2 (see Appendix for calculations). For a planar furanose ring the projected valency angles for gig hydrogens is 0° and for Eggng hydrogens is 120°. The only literature values for D-apiose containing compounds are those of Carey 33 2;! (1966) and Ball gt 22' (1968). Work- ing with the mono- and di-O-isopropylidene derivatives of D-apio-D-furanose and D-apio-L-furanose, having the d con- figuration at the anomeric carbon atom, these workers obtained values of 3.5-3.7 Hz for the apparent coupling constants between H-1 and H-2. They calculated that the dihedral angle between H-1 and H-2 is 40-500. Therefore the formation of the fused ring system in these is0pro- pylidene derivatives resulted in the D-apiose adopting the 115 "twist" conformation in which C-2 was below the plane formed by C-1, C-4 and the ring oxygen, and C-3 was above this plane (Hall, 1964). For apibiose phenylosotriazole, the value of 56:50 for the dihedral angle indicates that the linkage in api- biose has the a configuration and the non-reducing terminal furanose ring also has the "twist" conformation. 0n the other hand, the value of 121t5° for the dihedral angle indicates that the linkage has the 8 configuration and that this part of the furanose ring is planar. The entire ring is probably not planar however. Jardetzky (1960) has shown that the ribofuranose ring in nucleosides is not planar, The interaction between the two gig-eclipsed hydroxyl groups at C-2 and C-3 are such that the furanose ring adOpts a conformation to minimize this interaction. Results with carbohydrates in the furanose ring indicate they adopt the conformation that minimizes non-bonded interactions between adjacent substituents (Hall, 1964). Therefore, the config- uration of the linkage between the two D—apiose molecules of apibiose may be 8 and the non-reducing terminal U—apiose mOlecule has adopted one of the other possible conformations for the furanose ring (Hall, 1964). Therefore, the available data permit a tentative assignment of the 3 configuration for the anomeric linkage between the two D-apiose molecules of apibiose and the structure of the disaccharide is 3'-O-D-apio-D-furanosyl-D- 116 apiose (V). When attached as sidechains to the d-(1—94)— galacturonan, the disaccharide is (2 and/or 3)-O-[0-6-D- apio-D-furanosyl-(L—QB')-(a or S)-D-apio-(D or L)-furanosyl]- galacturonan. Additional data, such as the molecular rota- tions and proton magnetic resonance Spectra of the four methyl glycosides of D-apiose, is needed before this assign- ment can be substantiated. It had been postulated by Beck (1967) that the D- apiose in apiogalacturonans, isolated from I. minor, was present as monomeric sidechains. However, his conclusion was based on hydrolysis data alone. It is possible that re-examination of his polysaccharide preparations by the periodate oxidation and the mild hydrolysis procedures reported here, may reveal the presence of disaccharide units of D-apiose. There are a number of structural aspects of these apiogalacturonans which remain to be determined. These include the configuration at C-3 of the reducing terminal D-apiose of apibiose, the position and stereochemistry of the glycosidic linkage between apibiose and the galacturonan, determination of a repetitive sequences, if any, in the apiogalacturonans and molecular weight. These problems are now under study in preparation for investigating the biosyn- thesis of these polysaccharides. APPENDIX Calculation of Dihedral Angles With the Karplus Equation From Karplus (1959): J = J cos2 0 + K o J = observed coupling constant K = -0.28 Hz 0 = dihedral angle Jo = constant dependent on the quadrant of 0 From Abraham _§ 31. (in Hall, 1964): (A) Jo = 9.3 for 0° 3 0 a 90° (B) J0 .-_ 0.4 for 90°$¢ 3. 180° {b C... II 2.6 Hz JO = 9.3 0° g 0 s 90° 2.6 = 9.3 cos2 0 -0.28 cos2 0 = 0.31 cos 0 = 0.557 ¢=56° (B) J = 2.6 Hz J = 10.4 900 g 0 a 180° 0 2.6 = lOou' COS2 ¢ -O.28 ¢ = 121° The observed coupling constant is a time averaged constant since the molecule is in constant motion. Therefore the values calculated from the Karplus equation are only a time average approximation of the dihedral angle (Hall, 1964). 117 REFERENCES Albersheim, P. (1959). Biochem. biOphys. Res. Commun. 1, 253- Albersheim, P., Neukom, H. and Deuel, H. (1960). Arch. Biochem. BiOphys. 29, 46. ASpanill, G. 0. and Baillie, J. (1963). J. chem. Soc., p. 1702. ASpinall, G. O. and Fanshawe, R. S. (1961). J. chem. Soc., p. 4215. Bacon, J. S. D. (1963). Biochem. J. 82, 103P. Ball, D. H., Carey, F. A., Klundt, I. L. and Long, L., Jr. (1969). Carbohyd. Res. 19, 121. Bateman, D. F., Van Etten, H. D., EngliSh, P. D., Nevins, D. J. and Albersheim, P. (1969). Plant Physiol. 44, 641. Beck, E. (1966). Ber. dt. bot. Ges. Z], 396. Beck, E. (1967). Z. Pflanzenphysiol. 52, 444. Beck, E. and Kandler, 0. (1965). z. Naturf. 20b, 62. Bell, D. J., Isherwood, F. A. and Hardwick, N. E. (1954). J. chem. Soc., p. 3702. Bhattacharjee, S. S. and Timell, T. E. (1965). Can. J. Chem. fig, 758. Bouveng, H. 0. (1965). Acta chem. Scand. 12. 953. Bray, G. A. (1960). Analyt. Biochem. 1. 279. Carey, F. A., Ball, D. H. and Long, L., Jr. (1966). Carbohyd. Res. 2, 205. Dische, Z. (1962). In Methods in Carbohydrate Chemistry, vol. 1, p. 497. Ed. by Whistler, H. L. and Wolfrom, M. L. New York: Academic Press Inc. Duff, R. B. (1965). Biochem. J. 24, 768. 118 119 EnglishagP. D. and Albersheim, P. (1969). Plant Physiol. 217. __. Gauthier, P. B. and Kenyon, A. J. (1966). Biochem. biophy. Acta, 120, 551. Gorin, P. and Perlin, A. (1958). Can. J. Chem. 22, 480. Gupta, S. H. and Seshadri, T. R. (1952). Proc. Indian Acad. Sci., Section.A 22, 242, Gustine, D. L. and Kindel, P. K. (1969). J. biol. Chem. 244, 1382. Hakamori, S. (1964). J. Biochem. (Tokyo) 22, 205. Halford, H., Ball, D. H. and Long, L., Jr. (1968). Carbohyd. Res. 8, 363. Halford, H., Ball, D. H. and Long, L., Jr. (1969). Chem. Commun., p. 255. Hall, L. D. (1964). Adv. Carbohyd. Chem. $2, 51. Halyalker, H., Jones, J. K. N. and Perry, M. (1965). Can. J. Chem. 22, 2085. Hattori, S. and Imaseki, H. (1959). J. Am. chem. Soc. 81, 4424. Hemming, R. and Ollis, W. D. (1953). Chem. and Ind., p. 85. Imaseki, H. and Yamamoto, T. (1961). Arch. Biochem. Biophys. 22, 467. Jardetzky, C. (1960). J. Am. chem. Soc. 82, 220. KarpluS, M. (1959). J. Chem. Phys. 29, 11. Khalique, A. (1962). J. chem. Soc., p. 2515. Kindel, P. (1969). Carbohyd. Hes., in press, Klyne, H. (1950). Biochem. J. 32. p. xli. Lemieux, R. U. (1962). In Methods in Carbohydrate Chemistry, vol. 1, p. 45. Ed. by Whistler, R. L. and Wolfrom, M. L. New York: Academic Press Inc. Malhotra, A., Murti, V. V. S. and Seshadri, T. R. (1965). Tetrahedron Lett., p. 3191. 120 McComb, E. A. and McCready, H. M. (1952). Analyt. Chem. 33, 1630. Mendicino, J. and Picken, J. M. (1965). J. biol. Chem. 240. 2797- Morrison, R. T. and Boyd, R. N. (1966). Organic Chemistry, 2nd ed., p. 409, 982, 1019. Boston: Allyn and Bacon, Inc. Nakaoki, T.. Morita, N., Motosune, H., Hiraki, A. and Takeuchi, T. (1955). J. pharm. Soc. Japan.25, 171. Nelson, N. (1944). J. biol. Chem. $22. 375. Neukom, H. and Deuel, H. (1958). Chem. and Ind., p. 683. Norris, L., Norris, R. E. and Calvin, M. (1955). J. eXp. BOto Q. 6"". Ovodova, R. G., Vaskovsky, V. E. and Ovodov, Yu. S. (1968). Carbohyd. Res. 6, 328. Partridge, S. M. (1949). Nature, Lond. 164, 443. Rahman, A. “'Uo (1958). Z. Naturfo 12b, 201. Sandford, P. and Conrad, H. (1966). Biochemistry, 5, 1508. Schaffer, R. (1959). J. Am. chem. Soc. Q1, 5452. Schmidt, 0. T. (1930). Liebigs Ann. 483, 115. Schultz, T. H. (1965). In Methods in Carbohydrate Chemistr , vol. 5. p. 189. Ed. by Whistler, R. L. and Wolfrom, M. L. New York: Academic Press Inc. Silverstein, H. and Bassler, G. (1964). Spectrometric Identification of Organic Com ounds p. 71. New YSrk: John Wiley and Sons, Inc. ' Speck,J. 0., Jr. (1962). In Methods in Carbohydrate Chemistr , vol. 1, p. 441. Ed. by Whistler, R. L. and Wolfram, M. L. New York: Academic Press Inc. Trevelyan, W. E.. Procter, D. P. and Harrison, J. S. (1950). Nature, Lond., 166, 444. Vongerichten, E. (1901). Liebigs Ann. 218, 121. Vongerichten, E. (1902). Liebigs Ann. 22 . 71. 121 Vongerichten, E. and Mflller, Fr. (1906). Chem. Ber. 22. 235- Wagner, H. and Kirmayer, W. (1957). Naturwissenschaften 35+. 307. Weygand, F. and Schmiechen, H. (1959). Chem. Ber. 22, 535. Whistler, R. L. and BeMiller, J. N. (1958). Adv. Carbohyd. Chem. $2, 289. Whistler, H. L. and BeMiller, J. N. (1962). In Methods in Carbohydrate_§hemistry, vol. 1, p. 47. Ed. by Whistler, H. L. and Wolfrom, M. New York: Academic Press, Inc. Williamfié Dé T. and Jones, J. K. N. (1964). Can. J. Chem. __. 9- Zitko, V. and Bishop, C. T. (1965). Can. J. Chem. 22. 3206.