'EHE PLASTEIN REACTION

“tests €0,9- ‘x‘g‘m fieqvee ag M Sc

fi’ifiHEGAN SEATE UNEVERSITY
Thomas F. Diehl
1975

 

THESIS

 

 

ABSTRACT

THE PLASTEIN REACTION

BY

Thomas F. Diehl

Since 1886 the plastein reaction has been investi—
gated to determine if specific enzymes, under empirical
conditions, catalyze protein synthesis. Chymotrypsin is an
acyl transferase which normally transfers to water. When
water becomes limiting, chmotrypsin may transfer to free
N-terminal amino acids thus initiating protein synthesis
through a condensation-type reaction. A decrease in TCA-
solubility of plastein over that of the hydrolyzate, the
insolubility of plastein in solvents, the inability of
plastein to pass through dialysis membrane, and dramatic
changes in color and viscosity of the plastein over the
hydrolyzate have been cited by previous investigators as
evidence of peptide bond formation.

The ninhydrin reaction was used to monitor changes
in the number of N-terminals exposed. Column chromato—
graphy and disc gel electrophoresis were used to monitor
Changes in molecular weight profiles. These methods along

‘Vith others gave no indication of any increase in molecular

Thomas F. Diehl

weight of the plastein over the hydrolyzate. The changes
in physical properties of the hydrolyzates, such as
insolubility and molecular weight increases, were explained

on the basis of hydrophobic aggregation.

THE PLASTEIN REACTION

BY

Thomas F. Diehl

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Food Science and Human Nutrition

1975

ACKNOWLEDGMENTS

It is always hard to properly thank a friend who
has helped. Dr. Brunner has expressed personal interest
and enthusiasm for this project. His technical assistance
and philosophical guidance will always be remembered.
Sincere thanks to Mike Mangino for his consultation and
informative injectures of ideas. Ms. Ursula Koch made
possible the amino acid analysis and facilitated laboratory
bottlenecks. Thanks to my wife, Linda Kay, whose patience
and understanding has assured the completion of this
research.

In addition I wish to thank Dr. J. R. Kirk,
professor of food science, and Dr. D. R. Heldman, professor
of agriculture engineering, for serving on my guidance
committee.

Lastly, I wish to thank the Department of Food
Science and Human Nutrition for enabling me to continue my

education.

ii

TABLE OF CONTENTS
Page
LIST OF TABLES . . . . . . . . . . . . . . . . . . . vi

LIST OF FIGURES . . . . . . . . . . . . . . . . . . Vii

INTRODUCTION . . . . . . . . . . . . . . . . . . . . 1
LITERATURE REVIEW . . . . . . . . . . . . . . . . . 3
History . . . . . . . . . . . . . . . . . . . . 3
Preparation of Plastein . . . . . . . . . . . . 3
Hydrolysis . . . . . . . . . . . . . . . . . 5
Concentration . . . . . . . . . . . . . . . 5
Plastein Synthesis . . . . . . . . . . . . . 6
Proposed Mechanisms for Plastein Formation . . . 7
Cyclization . . . . . . . . . . . . . . . . 7
Condensation . . . . . . . . . . . . . . . . 7
Transpeptidation . . . . . . . . . . . . . . 8
Contemporatory Investigations . . . . . . . . . 10
Model Systems . . . . . . . . . . . . . . . 10

Gel Filtration . . . . . . . . . . . . . . . 11

Gel Electrophoresis . . . . . . . . . . . . ll

Amino Acid Analysis . . . . . . . . . . . . 12
EXPERIMENTAL PROCEDURES . . . . . . . . . . . . . . 13
Materials and Methods . . . . . . . . . . . . . 13
Preparation of Proteins . . . . . . . . . . 13

iii

Whole Casein . . . . . . . . . . . . .

Soy Protein Isolate . . . . . . . . .
Preparation of Plasteins . . . . . . . . .
Enzyme-Induced Plastein . . . . . . .

Enzyme Activity . . . . . . . . . . .

Heat Induced Plastein . . . . . . . .
Analytical Materials and Methods . . . . . . .
Amino Acid Analysis . . . . . . . . . . .
Incorporation of tyrosine ethyl ester

Column Chromatography . . . . . .'. . . .
Polyacrylamide Disc Gel Electrophoresis .

15% SDS-Phosphate System . . . . . . .

15% SDS-tris-glycine System . . . . .

Protein Identification . . . . . . . .
Ninhydrin . . . . . . . . . . . . . . . .
Nitrogen . . . . . . . . . . . . . . . . .
Recovery Methods . . . . . . . . . . . . .
Thin Layer Chromatography . . . . . . . .
RESULTS AND DISCUSSION . . . . . . . . . . . . . .
Enzyme Activities . . . . . . . . . . . . . .
Pepsin . . . . . . . . . . . . . . . . . .
a-Chymotrypsin . . . . . . . . . . . . . .
Plastein Recovery . . . . . . . . . . . . . .
Physical Characteristics . . . . . . . . .

Protein Recovery . . . . . . . . . . . . .

iv

Page
13
13
l4
14
15
16
16
16
18
18
19
20
20
20
22
23
23
24
25
25
25
25
28
28

29

Molecular Weights

Amino Acid Analyses

CONCLUSIONS

LITERATURE CITED

APPENDIX

Ninhydrin
Gel Filtration

Polyacrylamide Gel ElectrOphoresis

Hydrophobic Analyses

Incorporation of L-tyrosine Ethyl Ester

Page
31
31
33
33
40
40
44

52

54

58

Table

LIST OF TABLES
Page

Percent recovery of casein and soy plas-
teins from a 50% ethanol wash of the
reactants . . . . . . . . . . . . . . . . . 31

Units (mg) of protein required per unit
absorbance change in excess of a rea-
gent blank as determined by the nin-
hydrin assay . . . . . . . . . . . . . . . . 32

Amino acid analyses of casein hydroly—
zates and its plastein . . . . . . . . . . . 41

Amino acid analyses of soy hydrolyzates
and its plastein . . . . . . . . . . . . . . 42

Hydrophobicities of casein and soy
hydrolyzates and their derived
plasteins . . . . . . . . . . . . . . . . . 43

L—Tyrosine ethyl ester incorporation
into casein plastein . . . . . . . . . . . . 49

L-Tyrosine ethyl ester incorporation
into soy plastein . . . . . . . . . . . . . 50

vi

LIST OF FIGURES
Figure Page

1. Process for deodorizing, debittering and
synthesizing plastein (from Arai et a1.
1975) . . . . . . . . . . . . . . . . . . . 4

2. Proposed process for the reverse reaction
of a-chymotrypsin (from Arai et 31. 1975) . 9

3. Hydrolysis of casein (1% solution) with
pepsin (1:100 E/S) at 37 C (pH 7.0)
as monitored by the ninhydrin reaction . . . 26

4. Pictures of casein and soy plasteins and
their hydrolyzates (30% protein): casein
peptic hydrolyzate (A); heated casein
peptic hydrolyzate (B); casein plastein
using a-chymotrypsin (C); soy peptic
hydrolyzate (D); heated soy peptic
hydrolyzate (E); and soy plastein using
a-chymotrypsin (F) . . . . . . . . . . . . . 30

5. Gel filtration (Sephadex G-75) chromato-
gram of casein hydrolyzate (l) and casein
plastein (2) in tris-HCl buffer (1M NaCl) . 34

6. Gel filtration (Sephadex G-75) chromato-
gram of soy hydrolyzate (l) and soy
plastein (2) in tris-HCl buffer (1M NaCl) . 35

7. Electropherograms of dansylated casein
and soy with their plasteins in a 15%
Cyanogum-4l tris-glycine gel system:
Tube 1 & 2, heated soy peptic hydroly-
zate; tubes 3 & 4, heated casein peptic
hydrolyzate; tubes 5 & 6, peptic soy
hydrolyzate; tubes 7 & 8, peptic casein
hydrolyzate; tubes 9 & 10, casein
plastein; and tubes 11 & 12, soy plastein . 36

vii

Figure Page

8. Electropherograms of dansylated heated
soy plastein using a 15% Cyanogum-4l
gel in an SDS phosphate system: 50%
ethanol heated soy plastein pellet
(l & 2); 50% ethanol heated soy
plastein wash (3 & 4); and heated soy
hydrolyzate before the wash (5 & 6) . . . . 38

9. Effects of SDS on casein proteins and
its plasteins: whole casein in water
(1) and SDS (2); casein hydrolyzate in
water (3) and SDS (4); heated casein
hydrolyzate in water (5) and SDS (6);
and casein plastein in water (7) and
SDS(8).................. 45

10. Effects of SDS on soy proteins and its
plasteins: whole soy in water (1) and
SDS (2); soy peptic hydrolyzate in
water (3) and SDS (4); heated soy
hydrolyzate in water (5) and SDS (6);
and soy plastein in water (7) and
SDS (8) . . . . . . . . . . . . . . . . . . 46

11. Thin layer chromatogram of tyrosine and
its ethyl ester on silica gel plates
run with N-propanol and water (70:30
v/v) for three hours: position 1, an
equal mixture of 2 & 3; position 2,
L-tyrosine; and position 3, L-
tyrosine ethyl ester (HCl) . . . . . . . . . 47

viii

INTRODUCTION

The problem that concerned us was the nature of the
plastein reaction. There is abundant literature on this
topic with considerable conflicting conclusions.

A Japanese research team at the University of
Tokyo have devoted considerable effort since 1970 to the
investigation of the plastein reaction. They reported that
soy protein peptic hydrolyzate produced a white, tasteless
plastein. They reported that amino acids could be incor-
porated into plastein when corresponding ethyl esters were
added to the reaction mixture. Mixtures of complementary
protein hydrolyzates resulted in a plastein with a higher
P.E.R. than either of the original proteins.

Vegetable proteins have played an increasing role
in human nutrition and there is no indication that their
incorporation into food products will decrease. In an
effort to provide high quality, low cost vegetable proteins
with improved functional properties, the plastein reaction
was investigated.

The primary objective of this study was the

investigation of the claim that the plastein reaction is

a peptide bond forming reaction synthesizing polymeric
proteins from monomer peptides. The incorporation of amino
acids using their corresponding ethyl esters was also
investigated. From data obtained in these investigations

a suggested mechanism for plastein formation was proposed.

L ITERATURE REVIEW

History

In 1886 Danilewski observed the formation of a
precipitate when stomach extracts were added to a con-
centrated peptic hydrolyzate. Danilewski believed the
precipitate to be produced by the stomach enzymes since no
precipitate resulted if the stomach extracts were first
boiled. In 1895 Oknew confirmed these observations. In
1901 Sawjalow investigated the enzymatic phenomenon and
designated the resulting precipitate, "plastein." Wasteney
and Borsook (1930) published an informative review article

covering the early work on plastein.

Preparation of Plastein

 

The historical studies with plastein established
three conditions for plastein formation: (1) a peptic
hydrolyzate, (2) a sufficient concentration of the
hydrolyzate, and (3) a plastein-forming enzyme at the
correct pH and temperature, Fig. 1.

Traditionally, egg albumin was used as the sub—
strate for the plastein reaction (Horowitz and Haurowitz,

1959). Fujimaki gt 31. (1970) performed the plastein

Cmda wove-n mmentmhon
(I .wlum nq «I‘luut'vl‘s

m Launo {lot-3)

-- - nc'wnus - -'

 

I Hydm'y'us th eeropc-ghrfow

45‘ N (tune hydro’yscflc

A . ‘90 (confqmmq unpunhes
'S o0 :11 1'20. $0 1M)

°Ko c ./o —cc'c my: and Inflor—

l Tleaflmen! mm o'qomc when!

 

Residual 1! Iliad
Ls '
Puru'ved.hygr3l)sf: /‘ r . ‘ o how-hes
1 er > L ’ o .0 0. (odmann.
fun we)
a, 0 9 '
\— \ ‘ . .

Hydrolysn: mm empephdow / \ Synthesus mth cndopophdose

Hydrolysafe ' ‘ ' -' , /\ '
~14.” by“, bu! “ ' > ‘- {I \ I K Picslem 100-0") product
huvmg ammo cad- , . 'J \‘ L '\’ ---ahos!nuw<le~- -
Me taste-«- .. ' " \
a... / ‘ at

l Treafmcnt mth aqwom «banal

Rundue I lfimoct

/) I
Plastein ( g -| . \ chm MW
“no taste-— ( I Nphdes and

, ‘ p ‘ ‘ heedmmoocnds

 

FIGURE 1. PROCESS FOR DEODORIZING
AND DEBITTERING, AND FOR SYNTHESIZING
PLASTEIN (FROM ARAI EI.AL.. 1975).

reaction on a wide variety of plant and animal proteins.
Yeast, soy, and gluten protein hydrolyzates consistantly
gave the highest plastein yields. They described plastein
as a bland, white, protein-like substance possessing a

relatively high degree of insolubility.

Hydrolysis

The hydrolysis of egg albumin was usually achieved
by the addition of pepsin (Horowitz and Hoaurowitz, 1959).
Yamashita 33 31- (1970) reported enzyme/substrate ratios
of 1-3/100, employing a solution containing approximately
1% soy isolate at pH 1.8 for 24 h. This treatment resulted
in about 80% hydrolysis of the protein which Yamashita
considers optimum for plastein formation. Yamashita e: 31.
(1970) reported a molecular weight profile for the peptic
hydrolyzates of soy protein and the resulting plasteins by
means of gel filtration over Sephadex G-75. A significant
shift to higher molecular weight substances in the plastein
was noted. Tasi g£_al. (1974) observed that hydrolysis
fractions with average molecular weights between 685 and
1043 were more "plastein productive" than fractions above

or below this molecular weight range.

Concentration
Relatively high concentration of the peptic
hydrolyzate is essential for plastein formation. The con-
centration of the peptic hydrolyzate is reportly responsible

for the shift of enzyme "cleavage" properties to "peptide

r.

 

rs

bond forming" characteristics. Horowitz and Haurowitz
(1959) reported that 40% egg protein hydrolyzate was a
necessary condition for the plastein reaction. Tasi et a1.
(1972) reported that 6-8% soy peptic hydrolyzate was
necessary for plastein formation, but maximum plastein
yields occurred between 30-40% soy hydrolyzate. Concentra-
tion of the peptic hydrolyzate was usually accomplished
under vacuo at temperatures below 40 C. Tauber (1951b)
reported that extremely high peptide concentrations were
unnecessary if a 26% sodium chloride solution was used to
dilute the protein to 4% before subjection to the plastein

reaction.

Plastein Synthesis

Traditionally, plastein synthesis was catalyzed
with pepsin. Subsequently, selected plant extracts were
found to be "plastein active." Tauber (1951a) introduced
a-chymotrypsin as a plastein-forming enzyme. Pepsin
induces the formation of a precipitate as the plastein
reaction proceeds, whereas Chymotrypsin produces highly
viscious solutions. Fujimaki et_al. (1970) reported
excellent yields of plastein from soy isolate by employing
Chymotrypsin as the catalytic agent. None of a wide
variety of endopeptidases, ex0peptidases, and various
microbial proteases exceeded Chymotrypsin in plastein
productivity. Taminato et 31. (1972) prepared an insoluble

preparation of d-chymotrypsin bound to filterpaper. The

IA

 

r:

po.

5.

 

s .
«C

plastein reaction was then performed to dispell any
possibility that the plastein reaction is a product of
peptide-enzyme aggregation. Yamashita 32 31. (1971a)
reported plastein yields were a function of pH, concluding
that the pH optimum for Chymotrypsin shifts from around 7.0
for hydrolysis, to between pH 4 and 6 for optimum plastein

formation.
PrOposed Mechanisms for Plastein Formation

Cyclization
. Virtanem and Kerkkomem (1948) observed no increase
in molecular weight of plastein when compared to the peptic
hydrolyzate. They attributed the change in physical
properties of the hydrolyzate to cyclization of the pep-

tides.

Condensation

The condensation reaction catalyzes peptide bond
formation by the direct condensation of d-amino and a—
carboxyl groups. Determann 33 31. (1963) reported that in
high concentrations oligopeptides are converted to polymeric
forms. The monomers must be at least a tetrapeptide with
L-leucine, L-phenylalanine, or L-typosine as the C-terminal
residues. For example, when a pentapeptide with L-tyrosine
at the C—terminal and L-phenylalanine at the N-terminal, a
pentadecapeptide would be produced. Boyer (1971) discussed

the thermodynamics of the condensation reaction.

Transpeptidation

Transpeptidation reactions occur when enzymes
catalyze the transfer of acyl groups to water and other
acceptor molecules. Lehninger (1971) describes Chymotrypsin
as a hydrophobic acyl group transferase. Transpeptidations
are classified into two categories: (1) acyl transfer
(carboxyl transfer) and (2) imino transfer (amine transfer).
These reactions are catalyzed by esterases and the serine
proteases. Horowitz and Haurowitz (1959) believed that
amino acyl residues were transferred from donor peptides to
acceptor peptides or amino acids. Otherwise there was no
way to explain the constant amount of a-amino nitrogen
before and after the plastein reaction. Also, acceptor
peptides must be poor in threonine, asparatic, and glutamic
acids and rich in leucyl, isoleucyl residues to participate
in the plastein reaction. Thus, the insolubility of
plastein could be explained on the basis of the nonpolar
side chains of leucine, isoleucine, and phenylalanine.

Yamashita 33 31. (1973), using isotopic oxygen
(018) techniques, reports that transpeptidations occur via
acyl and imino transfer but that condensation reactions
seem to be the major mechanism involved in the plastein
reaction. Arai 33 31. (1975b) concluded that Ber-195 of
Chymotrypsin serves as the target of acyl-enzyme intermedi-
ate formation. In the reverse reaction, His-57 serves as
the acid-base catalytic site for acyl enzyme formation.

The proposed mechanism is presented in Figure 2.

A5 A3
___1 '0: -1 ”_ HE, _
C'O be
6 b
{I/‘J “ ’ 1‘1”
Hus ‘ N SM HI M N
51 (0:22 _ :95 SI (O7 w
‘N"'H -o ’N n -~o
015:? o c'----R
/ n ‘ /
H O H 3

His _
57

 

 

 

' _ _ I II
R—NH co R H 0

FIGURE 2. PROPOSED PROCESS FOR THE
REVERSE REACTION OF a-CHYMATRYFSIN
(FROM ARAI EI.AL.. 1975).

sh‘

in

10
ContemporaryAInvestigations

Model Systems

Determann and Wieland (1961) synthesized the
plastein active pentapeptide L-tyrosyl-L-isoleucyl-
glycyl-L-glutamyl—L-phenylalanine. A plastein was produced
from this pentapeptide using pepsin as the catalyst.
Utilizing UV spectrophotometry of the DNP-derivative, a
molecular weight of 2250 was determined for the plastein.
Subsequently, Determann 31 31. (1962) synthesized nine
plastein-active pentapeptides. The substrate plastein
mixture contained 100 mg of peptide in 0.1-0.2 m1 of water,
adjusted to pH 4 and incubated at 37 C for 15-24 h with
less than 1 mg of pepsin. The enzyme was destroyed by
heating to 80 C and plasteins were isolated by centri-
fugation. Plasteins formed by the use of pepsin possessed
low molecular weights and an average degree of polymeri-
zation of only 2.5.

Determann 3: 31. (1963) conducted experiments
designed to study the effect of chain length on the
plastein reaction. They found tetra-, penta-, and hexa-
peptides to be plastein-active while di- and tri-peptides
did not polymerize in the presence of pepsin. Aromatic
residues at the carboxyl terminal of the peptide appears
to be essential for the formation of a plastein. The
substitution of alanine for tyrosine at the N-terminal

position in the pentapeptide did not effect its ability to

11

form plasteins, but similar substitution at the carboxyl-
terminal caused inactivity of the peptide. Fujimaki
(1971) showed that peptic hydrolyzates of soy protein
effectively liberated bound odorants and lipids from the
native protein, however, the hydrolyzates possessed
extremely bitter flavor. Following the plastein reaction
the peptide mixture was completely void of bitterness.
Insolubility in aqueous systems is a characteristic of
plasteins. A50 33 31. (1973) obtained good solubility with
0.3 M sodium dodceyl sulfate (SDS) or 2N sodium hydroxide
(NaOH). They concluded that hydrophobic bonding was

largely responsible for the water insolubility of plastein.

Gel Filtration
Yamashita 3£_31, (1970) estimated the molecular
weight of soy hydrolyzate and soy plastein by gel filtration
over Sephadex, and observed that the average molecular
weight of the hydrolyzate increased by approximately three

fold following the plastein reaction.

Gel Electrophoresis
Tasi 33 31. (1974) published the results of
polyacrylamide gel electrophoresis in a phenol-acetic
acid-water (1:1:1) buffer system, indicating that the
‘molecular weight increased from 600—1000 to 11-27,000 as
a consequence of the plastein reaction. Ultracentrifugal
sedimentation analysis revealted an average molecular

weight of 20,000.

12

Amino Acid Analysis

Yamashita 33 31. (1970) reported amino acid
analyses for soy protein and soy plastein. A30 33 31.
(1974) showed that plasteins could be enriched with the
ethyl esters of specific amino acids. Yamashita 33 31.
(1974), using a diethyl ester preparation of glutamic acid,
reported a 17% increase in glutamic acid content of the
resulting plastein. The plastein was extensively washed
and dialyzed prior to assay. Amino acid incorporation is
of practical importance when one considers that proteins
of low nutritional quality can be improved by the addition
of limiting amino acid residues. Yamashita 33 31. (1971)
demonstrated that plasteins composed of hydrolyzates of
complementary proteins yielded higher P.E.R. values than

either of the original proteins.

EXPERIMENTAL PROCEDURES

Materials and Methods

 

Preparation of Proteins

Whole Casein

 

Fresh skim milk was adjusted to pH 4.6 with 6N HCl.
The precipitated casein was then filtered through three
layers of cheese cloth resuspended in water and adjusted to
pH 7.0 with 6N NaOH. The casein was acid precipitated and
resuspended in water two additional times before it was

shell frozen in preparation for freeze drying.

Soy Protein Isolate

 

Soy Promine-D isolate was donated by Central Soya
(Chicago, 111.). Promine-D is prepared from isoelectric
precipitated soybean proteins which were resuspended in
water at pH 7.0 and sprayed dried using low heat for maxi-
mum solubility. All protein preparations were stored in

air tight containers or plastic bags at 0 C.

13

14

Preparation of Plastein

Enzyme-Induced Plastein

 

A 1% solution of casein or soy protein was hydro-
lyzed at pH 1.6-2.0, using a pepsin-protein ratio of 1:100
(w/w) at 37 C for 24 H. The pepsin was 3X crystallized, a
preparation from Nutritional Biochemical Corporation. The
peptic protein hydrolyzate was neutralized to pH 7.0 using
6N sodium hydroxide (NaOH). Concentration of the peptic
hydrolyzate was carried out using a rotary evaporator at
40 C. A vacuum of 28 inches was produced with a mechanical
vacuum pump. The condensate was collected which enabled
estimation of protein concentration to between 30 and 35%
protein. After concentration, the pH was adjusted to 7.0
when necessary, and incubated with a-chymotrypsin at 37 C
at an enzyme:protein ratio of 2:100 (w/w). a—Chymotrypsin
was a salt-free, 3X crystallized preparation obtained from
Nutritional Biochemical Company. The reaction was allowed
to proceed for 24 h. The resulting plastein was washed
with a 100-fold excess of 50% ethanol and centrifuged at
1000 x g for 20 min. The white plastein pellet was sus—
pended in water, shell frozen, and 1yophi1ized on the
laboratory's freeze dryer (Arai 33 31., 1975b).

Plasteins were formed in dilute protein digest
solutions containing only 4% total solids and 26% sodium
chloride. The procedure of Tauber (1951b) was followed

with the following modifications. Soy hydrolyzate was

15

used as the substrate in place of egg albumin, the plastein
reaction was carried out at pH 7.0 instead of 7.3 and

sodium fluoride was omitted from the salt solution.

Enzyme Activity

 

The activity of a—chymotrypsin was determined by
the L-tyrosine ethyl ester method of Rick (1963). L-
tyrosine was purchased from Sigma Chemical Company and L-
tyrosine ethyl ester HCl (TEE) from Nutritional Biochemi—
cals Corporation. The a-chymotrypsin used was the same as
that used for the plastein reaction. All chemicals and
enzyme preparations were stored at 0C. Spectrophotometric
measurements were made with a Beckman DK-2A ratio-recording
spectrophotometer at a wavelength of 234 nm using silica
cuvettes with a light path of 1 cm. L-tyrosine was made
up to one millimolar in tris buffer (Appendix). A 2 mM
solution of tyrosine ethyl ester using the tris-buffer
plus 4.44 ml of a 5% CaCl2 was added per 100 m1 of solu-
tion. The CaCl2 was added to the L-tyrosine ethyl ester
solution to help stabilize Chymotrypsin. The enzyme solu-
tion contained 50 ug Chymotrypsin/.1 ml in tris-HCl buffer.
Three ml of a 1 mM L-tyrosine solution was used as a
reference in the DK-2A spectrophotometer. One and a half
milliliter of a 2 mM L-tyrosine ethyl ester solution was
diluted to 3 ml with enzyme and buffer. The decrease in

adsorbance was plotted against time. The activity of

l6

a-chymotrypsin was determined as follows:

A "3234/mln = u mole TEE hydrolyzed/

adsorbance 1 u mole .‘mg enzyme min/mg Chymotrypsin.
assay volume *

 

 

Chymotrypsin activity was determined according to
the method of Rich (1963) in sodium chloride solutions
(26%) with the modification that all solutions contained

26% sodium chloride (w/w) except the enzyme solution.

Heat-Induced Plastein

 

A 35% solution of casein or soy protein hydroly-
zate was prepared according to the method of Arai 33 31.
(1975a) and adjusted to pH 7.0 after hydrolysis and con-
centration. One to two ml portions of this solution were
placed in a test tube and then into a boiling water bath
for two min. The heat-induced plastein was washed with a
lOO-fold excess of 50% ethanol, centrifuged at 1000 x g

for 20 min and stored at 0 C for no more than 48 h.

Analytical Materials and Methods

 

Amino Acid Analysis
Amino acid analyses were performed on 22 h acid
hydrolyzates of protein samples (soy and casein), utilizing
the Beckman 120 C amino acid analyzer according to the
methods of Moore and Stein (1954), Moore 33_31. (1958), and
Spackman 33 31. (1958). Protein samples (2 mg/ml) were
weighed into a 10 m1 glass ampule and 5 ml of once-distilled

6 N HCl was added. A drop of octanol was added to reduce

F“.

3.
A:

l7

foaming when a problem. Each ampule was frozen in a dry-
ice ethanol bath under vacuum. The samples were slowly
thawed under vacuum to remove the dissolved oxygen. The
contents were again frozen and sealed under vacuum with an
air-prOpane flame. The sealed ampules were placed in an
oil bath at 110 C (i 0.1 C) for 22 h then removed and
allowed to cool. The ampules were broken and one milli—
liter of a 2.5 u mole/ml norleucine solution was added to
each sample as an internal standard. The contents of the
ampules were transferred to an evaporation flask connected
to a rotatory evaporator and evaporated to dryness at 40 C.
Samples were washed and dried until all traces of acid
were removed. Acid-free hydrolyzates were dissolved in
0.067 M sodium citrate-HCl buffer (pH 2.2), containing a
methionine antioxidant, thiodiglycol, BRIJ—35 (a detergent)
and the preservative, pentachlorophenol. Each solution
was transferred to a 5 ml volumetric flask and diluted to
volume with citrate buffer pH 2.2. Two tenths milli-
liter sample volumes were removed for analyses.

Using a FORTRAN program developed at Michigan State
University, samples were corrected for the internal
standard and column difference amino acid. The computer
program expressed the results in moles/1000 moles, gram

residue/100 gram sample and gram residue/100 gram protein.

18

Incorporation of Tyrosine
Ethyl Ester

 

 

Tyrosine ethyl ester-HCl was incorporated into soy
and casein hydrolyzate according to the method of A50
33 31. (1974) with the following modifications. Casein
and soy hydrolyzates were concentrated to 33% protein and
a 10 fold increase of tyrosine ethyl ester HCl was added.
The hydrolyzate was incubated at 37 C for 24 h. One gram
of the resulting plastein was washed with a lOO-fold
excess of 50% ethanol. Samples were centrifuged at 1000 x
g for 20 min., shell frozen and 1yophi1ized. A nitrogen
determination and amino acid analysis were performed on

the sample as previously described.

Column Chromatography

Casein and soy hydrolyzates were submitted to gel
filtration over Sephadex G-75. The sephadex was equili-
brated overnight in 0.1 M tris-HCl buffer containing 1 M
NaCl (see Appendix). The slurry was poured into the
column and after settling for 5 min the column outlet was
Opened to allow the gel bed to compress. The column was
equilibrated with four volumes of the tris-HCl buffer.
The void volume of the column was determined using Blue
Dextran 2000 and the column volume with B-mercaptoethanol.
The elutant was monitored with an Isco UV monitor at

280 nm.

19

Samples were dissolved in a SDS—phosphate buffer
(see Appendix) and heated for five min in a boiling water
bath. After cooling, 0.5 ml of a 0.5% protein solution
was placed on the column by removing the running buffer
above the column, layering the sample on top, and allowing
the sample to run into the bed, followed with 2-3 ml of

running buffer before continuing elution.

Polyacrylamide Disc Gel Electrophoresis

A stock solution of Cyanogum 41 was diluted and
polymerized with 0.25 ml of a 1.5% ammonium persulfate
solution and 50 pl of TEMED (see Appendix). Water was
carefully layered on the gels cast in 8 cm long glass
tubes with an outside diameter of 8 mm. The overlayer of
water was removed after polymerization and the gels were
placed in a Buchler lZ-hole or in a laboratory constructed
6-hole, electrophoresis apparatus. The upper and lower
buffer reservoir were filled with the appropriate running
buffer and electrophoresed at 8—10 mA/tube using a Bio-
Rad (model 400) or a Heathkit variable voltage power
supply. All samples contained bromphenol blue as the
marker dye. ElectrOphoresis in the presence of SDS was
performed according to Weber and Osborn (1969). Fluores-
cent SDS electrophoresis and fluorescent photography was

performed according to Talbot and thantis (1971).

20

15% SDS-Phosphate System

 

A 30% Cyanogum 41 stock solution was diluted 1:1
with 0.2 M phosphate gel buffer. The gel buffer was
diluted 1:3 to yield a 0.05 M SDS running buffer (see
Appendix). Electrophoresis was performed for 4—5 h at

room temperature with 8-10 mA/tube.

15% SDS-Tris-G1ycine System

 

A 15% Cyanogum 41 gel, containing tris was cast
according to the method of Subbaih and Thompson (1974).
The running buffer contained 25 mM tris-glycine (pH 8.3)
and 0.1% SDS (see Appendix). Protein samples were
dansylated according to the method of Talbot and thantis
(1971), and electrophorsed for 4-5 h at room temperature.
After electrophoresis, the position of the marker dye was
marked on the glass tube with a fluorescent crayon and

photographed with Polaroid type 57 high speed film.

Protein Identification

 

Protein (0.1-1.0%) solutions were prepared from
freeze—dried preparations and dissolved in a 0.05 M sodium
phosphate (NaZHPO4) buffer, pH 8.2. SDS was added to the
protein solution in the ratio of 2:1 (SDS/protein). Twenty
microliters of a 10% dansyl chloride in acetone solution
was added per milliliter of protein solution with vigorous
shaking. The mixture was placed in a boiling water bath

for five minutes. After cooling, sucrose was added to

21

increase sample density. The dansylated protein was layered
on a Bio-Gel P-6 column with an exclusion limit of 4,600
daltons to remove free dansyl. The Bio-Gel P-6 column had

a bed volume of 46 ml, measured 26 cm in length, and was
equilibrated with a 0.05 M phosphate buffer (pH 7.1) con-
taining 1% SDS. The void volumes and column volumes were
collected for electrophoresis.

Ultraviolet illumentation of dansyl (l-dimethyl-
amino-S-naphthalene—sulfonyl chloride) was generated with
a Black Ray UVL-21 with a peak output at 366 nm. The
separation of free dansyl from proteins could be readily
observed on the Bio-Gel P-6 column. Visual separation of
proteins could be observed during electrophoresis by
placing the UV source near the gels. Marker dye (brom-
phenol blue) was added to all protein samples before
electrophoresis. Using a fluorescent crayon manufactured
by Ultra-Violet Products (C-138 invisible chartreause),
the position of the marker dye could be located on the gel
tubes in the photograph.

Pictures of the fluorescent gels (15%) were taken
with a 4" by 5" press-type camera equipped with a Polaroid
single sheet adaptor. Polaroid type 57 (ASA 3200) film
was used in combination with a Kodak Wratten gelatin filter
No. 15 (yellow). The UVL-21 Black Ray ultra-violet light

was suspended above the gels placed on a Plexiglass plate.

22

Exposure times were for one second at f4.6 to f8.0 depend-
ing on protein concentrations.

Molecular weight estimations were made from a plot
of the relative mobilities vs. the log of molecular weights
of the protein standards. The standards used were oval-
bumin (45,000 daltons), Chymotrypsin (25,000 daltons), and
ribonuclease (13,700 daltons). Talbot and thantis (1971)
reported that relative mobilities calculated from photo-
graphs of fluorescent gels gave accurate and reproducible
molecular weight estimations. Relative mobilities were
calculated from measurements of the protein migration
zones and dye migration distance from the following rela—
tionship: R. M. (relative mobility) = distance protein

migrated/distance dye migrated.

Ninhydrin

Changes in a—amino nitrogen were monitored with
the ninhydrin assay. Protein solutions (0.1 m1 of a 0.1%
concentration) were adjusted to 0.5 ml by the addition of
distilled water and 1.5 m1 ninhydrin solution added (see
.Appendix). The mixture was heated in a boiling water bath
for 20 min and cooled to room temperature. Eight milli-
liters of a 50% n-propanol solution was mixed with this
solution and allowed to stand 10 min for color development.
.Absorbance was measured at 570 nm against a reagent blank.
Casein and soy hydrolyzates were used to prepare a standard

curve according to a procedure described by Clark (1964).

23

Nitrogen

Duplicate nitrogen analyses were determined by
means of a micro-Kejldahl apparatus. Ten to twenty milli-
grams of dried sample were mixed with 4 m1 of digestion
mixture (see Appendix). Digestion was carried on for 1 h.
The contents of the digestion flasks were allowed to cool
for 30 min and 1 ml of 30% H202 was added. Digestion was
continued for another hour and allowed to cool for 30 min.
The flasks were rinsed with 10 ml of deionized water and
allowed to cool for an additional 30 min. The mixture was
neutralized with 25 ml of a 40% NaOH solution and the
released ammonia steam distilled into 15 m1 of 4% boric
acid solution containing 5 drops of indicator solution
(see Appendix). Distillation continued until a final
volume of 75 ml was obtained. The ammonia-borate complex
was titrated with a 0.020 N HCl solution standardized with
tris-hydroxymethylaminomethane. Volumes from a blank were

substracted from sample titration values.

Recovery Methods
The plastein reaction was performed with both soy
and casein hydrolyzate concentrates at 33% concentration
and washed with lOO-fold quantities of 50% ethanol then
shell frozen in a dry ice-ethanol bath and 1yophilized.
The hydrolyzates were weighed and multiplied by their
nitrogen value. This value divided by the initial weight

gave the plastein yield.

24

Thin Layer Chromatography

Thin layer chromatography (TLC) was used to deter-
mine the purity of L-tyrosine ethyl ester HCl (TEE) and
L-tyrosine. TLC was performed according to a procedure
described by Brenner and Niederwiser (1967).

In the presence of tyrosine or cystine, 0.1 M HCl
was used to dissolve samples since these amino acids are
only sparingly soluble in other solvents. Prepoured silica
gel (Merck) plates, 20 by 20 cm and 250 pm thick, were
activated in a forced air oven at 110 C for 10 min. Ten
microliters of a 0.1% solution of TEE and tyrosine were
spotted and dried with a hair dryer. After the samples
were spotted, the plates were run unidimensionally in a
chromatographic chamber equilibrated with n-propanol and
water (70:30 v/v) at room temperature for 3 h. The silica
plates were removed from the chromatographic chamber and
air dried, then sprayed with a ninhydrin spray (see
Appendix). After staining, the plates were air dried for

4 min at 110 C to achieve visible zones.

RESULTS AND DISCUSSION

Enzymatic Activities

 

Pepsin

The ninhydrin test was used to evaluate the extent
of hydrolysis of casein by pepsin. Ninhydrin reacts
stoichrometrically with free amino groups and the color
produced is measured at 570 nm. Hydrolysis can be esti-
mated by substracting initial values for the sample from
values for the hydrolyzate (see Appendix). Data presented
in Figure 3 illustrate that casein was approximately 70%
hydrolyzed when exposed to pepsin at 37 C for 24 h. Most
of the hydrolysis occurred in the first three hours of
digestion. Using 25,000 as an average molecular weight
for casein, 70% hydrolysis should produce peptides with a
molecular weight of about 7,500. For a more accurate
determination of the number of peptide bonds hydrolyzed,
Whitaker (1972) recommends that the sample be subjected to
reduced alkylation followed by the addition of trinitro-

benzene sulfonic acid and observed at 570 nm.

a-Chymotrypsin
L-tyrosine ethyl ester (HCl) was used as a model
substrate to calculate an activity of 0.4 um/min/mg for

25

26

 

 

 

100-
80L-
§ 50A-
g
N
40—
20
I I J l l l J
0 1 2 1 r 23 24 25
TIME (H)

FIGURE 3. HYDROLYSIS OF CASEIN (1% SOLUTION) WITH
PEPSIN (1:100 E/S) AT 37 C (PH 7.0) AS MONITORED BY
THE NINHYDRIN REACTION.

27

a-chymotrypsin at pH 7.0 and 25 C. Tauber (1951b) reported
that substances similar to plasteins were formed when
diluted protein digests (4%) in saturated NaCl (26%) solu-
tions were reacted with a-chymotrypsin. In this study the
activity of a-chymotrypsin in a saturated NaCl solution

was 1.4 uM/min/mg at pH 7.0 and 25 C. It appears that in
the presence of NaCl the activity of a-chymotrypsin
increased about 3 fold. Turbidity can interfere with
absorbance at low wavelengths (234 nm). Increase in
turbidity was monitored at 600 nm. Thus, it was determined
that the decrease in adsorbance was due to turbidity and
that a—chymotrypsin was active in high salt solutions.

A 4% solution of L-tyrosine ethyl ester HCl in
tris buffer, pH 7.0, containing 26% NaCl was monitored for
changes in turbidity at 600 nm following the addition of
a-chymotrypsin. After 3.26 h, absorbance increased by
0.36 units above that of the control and the solution was
visibly turbid. Thus, as L-tyrosine ethyl ester is cleaved
in high salt solutions, tyrosine associated and precipitated
out of solution. Because tyrosine is a hydrophobic amino
acid, aggregation occurs through hydrophobic forces.
Brenner and Wiederwiser (1967) recognized the insolubility
of tyrosine in buffer solutions and suggested that 0.1 N
HCl be used as a solvent. Apparently the high salt con-

centration (26%) favors the aggregation of free tyrosine.

28

Tauber (1951b) reported that after 3 h at 37 C the
peptic hydrolyzate solution (4%) in 26% NaCl became turbid
following the addition of a-chymotrypsin, and after 24 h a
precipitate formed. This observation was duplicated with
soy hydrolyzate and the results correlated with those
observed by Tauber. Additionally, it was observed that a
heavy precipitate formed if the soy peptic hydrolyzate was
heated for 5 min in a boiling water bath. Neurath (1963)
reported that short polypeptide helices without side—chain
interactions have only marginal stability in solution.
Thus, if the salt solution depresses ionic side chain
interactions, only hydrogen and hydrophobic side chain
interactions remain to stabilize the system. The fact that
.heat above 60 C destabilizes hydrophobic interactions
(Neurath 1963) and that a-chymotrypsin cleaves aromatic
annino acids at the C-terminal residue suggests that aggre—

<gates are formed by hydrophobic interactions.
Plastein Recovery

Physical Characteristics
Soy hydrolyzate concentrate (30%) is turbid dark
brtnmn in appearance. Following the plastein reaction, the
runirolyzate changes color to a light tan. Casein hydroly-
zaixa concentrate (30%) was opaque white with a pinkish
caEN:. Following the plastein reaction, the protein con-

cenrtrate changes in appearance to a chalk white (see

29

Figure 4). A dramatic increase in the viscosity of the
plastein solutions was noted with soy and casein hydroly-
zates. Soy hydrolyzate is extremely bitter, even in dilute
solutions. Following the plastein reaction the taste is
essentially bland. After washing the plasteins with 50%
aqueous ethanol and centrifuging at 1000 x g for 20 min a

snow white pellet was obtained.

Protein Recovery

A 50% ethanol wash was used to separate the plas-
tein from lower molecular weight peptides and free amino
acids. Both ninhydrin and Kjeldahl nitrogen assays were
employed to estimate the percentage of protein in the
ethanol wash and pellet (see Table l). Plastein yields
from soy and casein hydrolyzates did not exceed 30% and 7%
respectively.

Fujimaki 33 31. (1970) expressed plastein produc-
tivity as the percentage of protein which is insoluble in
10% TCA. This TCA insoluble protein is collected by
centrifugation and washed with 50% ethanol to remove bitter
peptides. Whitaker (1972) reported that the change in
TCA.solubility of a protein-containing system is the method
most.widely used for monitoring hydrolysis of a protein.
Because the amount of material soluble in TCA decreased
instead of increasing during the plastein reaction,
Fujimaki 33_31, (1971) assumed that synthesis had occurred.

He reported that 95% of soy hydrolyzate and 48% of casein

30

 

 

FIGURE 4. PICTURES OF CASEIN AND SOY PLASTEINS AND THEIR
HYDROLYZATES (30% PROTEIN): CASEIN PEPTIC HYDROLYZATE (A):
HEATED CASEIN PEPTIC HYDROLYZATE (B); CASEIN PLASTEIN

USING “-CHYMOTRYPSIN (C); SOY PEPTIC HYDROLYZRTE (I);

HEATED SOY PEPTIC HYDROLYZATE (E); AND SOY PLASTEIN USING

a—CHYMOTRYPSIN (F).

31

Table 1.-—Percent recovery of casein and soy plasteins
from a 50% ethanol wash of the reactants.

 

 

 

 

Sam 1e % Protein in pellet % Protein in wash
p Ninhydrin Kjeldahl Ninhydrin Kjeldahl

Casein
hydrolyzate 0'0 _- 100 —_
Casein
plastein 5.88 6.50 94.12 93.5
Heated
Casein a 0.0 0.0 100 100
hydrolyzate
Soy __ __
hydrolyzate 5'8 94'2
Soy plastein 27.0 27.0 73.0 73.0
Eigfigiyigze 14.3 17.0 85.7 83.0

 

aHeated casein and soy hydrolyzates for 5 min at 100 C.

hydrolyzate were converted to plastein. Compared with
results obtained in the present study, these values repre-
sent a difference of 72% and 86% yield for soy and

casein plastein, respectively. A feasible explanation
might be that much of the protein which is insoluble in
TCA becomes soluble in aqueous ethanol. Thus, the yield

of plastein from these sources need further study.

Molecular Weights

 

Ninhydrin
Whitaker (1972) reported that ninhydrin reacts

stoichiometrically with free amino groups which can be

32

related to the number of peptide bonds hydrolyzed. As
enzymatic hydrolysis proceeds, more color is generated
because more amino groups are exposed per unit of protein
(see Table 2). If the plastein reaction were a simple
condensation or cyclization reaction, a decrease in the
number of free amino groups would be expected, and a cor-
responding increase in units of protein per absorbance
unit. The results of this study indicate that the reverse
situation is the case. An increase in the number of free
amino groups is concluded because of a decrease in units
of protein per absorbance unit. This indicates additional
hydrolysis of the protein system when a—chymotrypsin is
added.

Table 2.--Units (mg) of protein required per unit absorbance

change in excess of a reagent blank as determined
by the ninhydrin assay.

 

Sample mg/Abs
Casein protein 1.05
Casein peptic hydrolyzate 0.44
Heated casein peptic hydrolyzatea 0.40
Casein plastein 0.33
Soy protein 1.06
Soy peptic hydrolyzate 0.42
Heated soy peptic hydrolyzatea ' 0.40
Soy plastein ' 0.30

##

aHeated casein and soy hydrolyzates for 5 min at 100 C.

33

Gel Filtration

Sephadex G-75 produced elution patterns for casein
and soy hydrolyzates and their corresponding plasteins (see
Figures 5 and 6). These elution patterns demonstrated no
evidence that there was significant increases in the
molecular size of the plasteins. The disappearance of the
void volume elution peaks and the increases in volumes for
the slower eluting peaks suggest that hydrolysis continued
during the plastein reaction. This observation contradicts
those reported by Yamashita 33 31. (1970) who demonstrated,
using Sephadex-gel filtration, a significant shift to
higher molecular weight species after the plastein reaction.
They used a standard buffer proposed by Wolf and Briggs
(1956) which does not contain the high salt concentration
nor was the protein sample treated with SDS before being
applied to the column. Thus, the increase in molecular
weight of plasteins observed by Yamashita was probably due

to hydrophobic effects.

Polyacrylamide Gel Electrophoresis
Casein and soy hydrolyzates were dansylated and
electrophoresed in 15% Cyanogum—41 polyacrylamide gels
using either a tris-glycine or phosphate buffer system.
Figure 7 illustrates that soy hydrolyzate consists of two
major zones. A zone of approximately 90,000 daltons or
greater and a wide zone with an average molecular weight

of about 10,000. Casein digest contains only a single

34

.Aqu<z EHV mmmmnm JUIImeh zm ANV zmmkw<4m szw<u Qz<
AHV mh<N>JOmQ>I z_mw<u mo z<m00H<zoqu Ammlw xmo<zmmmv zouh<mb4_u 4mm .m mmscfim

Aazv mzsso> zoahssm

 

 

 

 

 

oma OOH cm 0
a a L
11/ II ‘3‘
23.1.“: / \
25.13555-.. xix _
1

 

 

ooom z<Exmo Him Y 1

BSNOdSBH 33080333

35

.3042 2.: mmHEDm .6112”: zH ANV zumhm<4m >om Qz<
A: mh<N>Jomo>I >ow no z<m00k<zomxu Ammlo wa<Immmv on._.<m._...:n_ 4mm .w mngu—

ADZV mzsso> zoapssm
CE 02 am

 

_ _ a

 

 

 

AINU

\
25.1.“: X x

.‘

zawa>mkoz>zo-u ,, \\

‘

BSNOdSEH 33030033

 

Doom z<mhxmo mssm \

~
\
V

36

 

FIGURE 7. ELECTROPHEROGRAMS 0F DANSYLATED CASEIN AND SOY
WITH THEIR PLASTEINS IN A 152 CYANOGUM-41 TRIS-GLYCINE GEL
SYSTEM: TUBE 1 8 2, HEATED SOY PEPTIC HYDROLYZATE; TUBES 3
a 4, HEATED CASEIN PEPTIC HYDROLYZATE; TUBES 5 8 E, PEPTIC
SOY HYDROLYZATE; TUBES 7 8 8, PEPTIC CASEIN HYDROLYZATE;

TUBES 9 8 10, CASEIN PLASTEIN; AND TUBES 11 8 12, SOY
PLASTEIN.

37

band with an average molecular weight of about 9,000.
Heating the soy and the casein hydrolyzates had no effect
on the electrophoretic patterns (see Figure 7). Similarly,
the casein and soy plasteins showed no increase in molec-
ular weight. The two zones located above the low molecular
weight in the casein plastein (patterns 9 and 10) have
molecular weights of 25,000 and 12,500 and were attributed
to hydrolytic production of a-chymotrypsin. In the case
of soy plastein only the 12,500 band was observed above
the lower molecular weight zone.

The concentration of lower molecular weight pro-
teins in the 50% aqueous ethanol wash of heated soy
hydrolyzate was obvious when heated soy hydrolyzate was
compared to the pellet and the decanted supernatant (see
Figure 8).

These results demonstrate that there was no
increase in molecular weight of the plastein over the
hydrolyzate. The 50% ethanol wash extracts the larger
jpolypeptides which were not hydrolyzed by pepsin. These
results are contrary to those of Tasi 33 31. (1974) and
.Arai 33 31. (1975) who used 7.5% polyacrylamide gel and a
phenol-acetic acid-water system and demonstrated an
increase in molecular weight of the plastein over the
peptic hydrolyzate. Mangino (1972) concluded that a
phenol—acetic acid-water system is not sufficient to dis-

sociate hydrophobic bonds of membrane proteins, thus Tasi

38

 

FIGURE 8. ELECTROPHEROGRAMS 0F DANSYLATED HEATED SOY
PLASTEIN USING A 15% CYANoeum-Ul GEL IN AN SDS PHOSPHATE
SYSTEM: 50% ETHANOL HEATED SOY PLASTEIN PELLET (1 8 2);
50% ETHANOL HEATED SOY PLASTEIN NASH (3 8 4); AND HEATED
SOY HYDROLYZATE BEFORE THE WASH (5 8 6).

39

and Arai may well have observed the hydrophobic aggregation
of polypeptides.

Castimpoolas 33 31. (1968) followed the natural
degradation of soy globulins during seed germination with
7% polyacrylamide gel using a tris-glycine discontinuous
buffer system. They observed a high molecular weight
protein zone that failed to show indications of proteolysis
after 16 days of germination. Thus certain fractions of
the soy globulin appear resistant to enzymatic hydrolysis.

In this current study, protein hydrolyzates were
dansylated because of difficulty in staining the electro-
phoretically resolved zones. Tasi 33 31. (1974) and Arai
33 31. (1975) also experienced difficulty in staining
hydrolyzed proteins. Talbot and thantis (1969) reported
that as molecular weight of the enzymatic substrate
decreased, the binding capacity of napthal blue black for
the proteinaceous products also decreased. Swank and
Munkres (1971) theorized that low molecular weight peptides
were leached from the gel by the staining solution. Dansy—
1ation overcomes these problems since the gels are never
removed from the tubes in which they were cast and electro-
phoresed. The identification of protein zones was
increased 100-fold over napthal blue black (Talbot &
thantis 1969).

Swank and Munkres (1971) proposed a disc electro—

phoretic method for analysis of oligopeptides in the range

40

of 8,500-1,800 daltons. Their procedure coupled with an
improved dansylation procedure, i.e., eliminating the
formation of dansylhydroxide, would perhaps allow for the
location of the 2250 dalton plastein monomer reported by
Determann and Wieland (1961). The value of sodium dodecyl
sulfate (SDS) as an effective dissociating agent for
apolar associated species of such low molecular weight is
questionable. Results obtained in this study indicate that
a low molecular weight compound such as dansylated L-
tyrosine ethyl ester not only appeared in the void volume
of a P-lO column but demonstrated lower electrophoretic

mobility than expected on the basis of size.

Amino Acid Analyses

 

Hydrophobic Analyses

Amino acid analyses were performed on 22 h HCl
hydrolyzates of casein and soy protein and their corres-
ponding plasteins (see Tables 3 and 4). Bigelow (1967)
proposed a method for calculating the average hydropho-
bicity of a protein from its amino acid composition.
Tanford (1962) defined the hydrOphobicity of an amino acid
based on the free energy of transferring one mole from an
aqueous solution at a fixed concentration to an ethanolic
solution at the same concentration. Transfer free energies
(HO) for side chains were calculated in calories/residue

by substracting the free energy change of glycine from each

41

Table 3.--Amino acid analyses of casein hydrolyzates and
its plastein.

 

 

 

acigmiggid Referenge Casein samplesb A
hydrolyzed) values Hydrolyzate Plastein Heated”
Ala 2.39 2.73 3.68 2.71
Arg 3.67 3.90 3.40 3.32
Asp 6.13 7.14 6.54 6.98
Cys/2 0.28 0.00 1.82 0.00
Glu 19.63 22.53 9.92 22.17
Gly 2.05 1.70 3.29 1.67
His 2.74 3.00 2.39 2.64
Ile 5.26 5.21 4.83 5.19
Leu 7.93 5.58 13.30 8.86
Lys 7.18 8.26 5.81 7.64
Met 2.46 0.80 1.19 1.78
Phe 4.45 5.29 7.56 5.04
Pro 9.53 11.79 9.24 10.73
’Ser 5.22 5.54 6.01 5.49
Thr 4.15 4.10 5.20 4.12
Trpe 1.70 ---- ---- ----
Tyr 5.67 5.79 7.27 5.45
Val 6.09 6.64 8.47 6.20

 

aLiterature values: Gordon 33_31. (1949).
Grams amino acid residues/100 grams protein.
CHeated casein hydrolyzate for 5 min at 100 C.

eNot determined.

42

Table 4.—-Amino acid analyses of soy hydrolyzates and its
plastein.e

 

 

 

Amino Reference Soy samplesC
Egégoiggig) avalues b Hygigly- Plastein Heatedd
Ala 4.08 3.60 3.80 3.93 3.76
Arg 7.45 7.55 7.57 7.98 9.58
Asp 11.51 10.38 12.16 10.30 11.80
Cys/2 1.78 1.34 0.50 0.73 0.23
Glu 16.94 18.42 20.65 13.76 15.15
Gly 4.88 3.44 3.71 3.38 3.00
His 2.66 2.25 2.43 2.13 2.09
Ile 5.20 4.40 4.80 7.07 5.73
Leu 6.73 6.66 8.30 11.02 10.31
Lys 5.81 6.01 6.19 6.54 7.10
Met 1.25 1.37 0.62 0.33 0.21
Phe 4.29 4.46 5.75 8.11 7.79
Pro 6.27 5.30 5.89 4.48 4.88
Ser 5.45 4.61 5.25 5.29 5.78
Thr 3.58 3.66 3.78 3.88 3.35
Trp 1.34 1.17 ———- ---— --—-
Tyr 3.34 3.51 3.76 4.77 4.12
Val 4.97 4.55 4.84 6.30 5.14
aLiterature values: Yamashita 33 31. (1971b) g A.A./100 g
protein.
Literature values: Rackis 33 31. (1961) g A.A./100 g
protein.

Grams amino acid residues/100 grams protein.
Heated soy hydrolyzate for 5 min at 100 C.
Not determined.

43

amino acid (see Appendix). Average hydrophobicities cal-
culated for soy and casein hydrolyzates and their plasteins
are presented in Table 5. These data indicate an increase
of 220 calories/residue for the plasteins over their
respective hydrolyzates. This increase in average hydro-
phobicity for the protein can only arise from a relative
increase in hydrophobic amino acids. The increase in
free energy of transfer was larger for heated soy hydroly-
zate than for heated casein hydrolyzate, indicating that
hydrophobic amino acid residues participate to a greater
extent in stabilizing soy.

As protein becomes denatured, an increase in
turbidity is frequently observed. SDS is an excellent
anionic detergent which disassociates hydrophobic bonds

Table 5.--Hydrophobicities of casein and soy hydrolyzates
and their derived plasteins.

 

 

HO . a
Sample cal/residue th'

casein hydrolyzate 1,215 1,215
casein plastein 1,435 ___
heated casein hydrolyzate 1,236 __—
soy hydrolyzate 1,044 _--
soy plastein 1,254 ___
heated soy hydrolyzate 1,181 -——

 

aValue of a—casein reported by Bigelow (1967).

44

(Reynolds & Tanford, 1970). Smith and Circle (1972)
report that secondary and tertiary structures of the major
globular components of soy proteins appear to be composed
of both random and compactly folded regions stabilized by
hydrophobic bonds. Casein proteins assume a random-like
structure in solution. Figure 9 illustrates that the
solubility of casein is only slightly affected by SDS and
that its hydrolysis by pepsin does not increase its solu-
bility. Heating the casein hydrolyzate produced a slight
amount of insoluble material which was quickly resolubilized
by the addition of SDS. Notice the significant increase in
protein insolubility which results from the plastein
reaction. This high level of insolubility was reduced by
the addition of SDS, indicating the strong contribution of
hydrophobic effects.

Soy proteins reflect their increased sensitivity
to SDS (Figure 10) which was expected because the protein
structure is substantially stabilized by hydrophobic bonds.
Because SDS is capable of solubilizing heat- and enzyme—
induced destabilization effects, it appears that these

aggregates are essentially hydrophobic in nature.

Incorporation of L-Tyrosine Ethyl Ester
The purities of L—tyrosine ethyl ester and L-
tyrosine were determined by thin layer chromatography
(Figure 11). L-tyrosine ethyl ester contained traces of

free tyrosine. L—tyrosine contained significant impurities

45

.Amv mom az< ARV mmh<z zs zampm<33
z_mm<u az< «Amy mom az< Amv mmp<z za mA<N>30ma>z zamm<u omh<mz «Aqv mam

az< Amy mmk<z za mA<N>Domo>I zamw<u AANV mom az< AHV mmh<z za zsmm<o
NDOIz “mzfim»m<3¢ mp“ az< mzampomm z_mm<o zo mam lo mhummum .m mm=o_¢

L0
L0

 

 

 

 

 

...................IIHII2
mm
"TIMI:
”H.111:

 

(NN 009) BONVBBOSEV

46

. .Amv mam az< ARV mmp<z za
szAm<3a >ow az< «Amy mam oz< Amv mmh<z z“ mp<~>30¢g>z >om awk<mz «Any

mam az< Amv mwA<z za mk<~>3oma>z u_Aamm >om «ANV mam n24 AHV mmk<z za
>om m3013 "mzzmhm<3a aha az< mZHNAomE >om zo mam mo whammmm .OH mmonm

00
l\
LO
Ln

3 m

N
H

 

 

 

 

 

I.

...............4

 

(WN 00g) BONVBBOSBV

47

 

 

 

 

 

 

 

 

FIGURE 11. THIN LAYER CHROMATOGRAM 0F TYROSINE AND
ITS ETHYL ESTER ON SILICA GEL PLATES RUN WITH N-
PROPANOL AND WATER (70:30 v/v) FOR THREE HOURS:
POSITION 1, AN EOUAL MIXTURE 0F 2 8 3; POSITION 2.

L-TYROSINE; AND POSITION 3, L-TYROSINE ETHYL ESTER
(HCL).

48

as evident from the trailing zone. 'An acid hydrolyzate of
L-tyrosine ethyl ester was compared to an unhydrolyzed
sample by amino acid analysis. Approximately 10% of the
tyrosine ethyl ester was free tyrosine.

A ten-fold excess of tyrosine ethyl ester was
added to the casein hydrolyzate (600 mg TEE/g protein).
The plastein reaction was initiated and after an ethanol
wash (1:100 v/v) the plastein pellet was analyzed for its
amino acid composition. Almost complete incorporation of
the tyrosine into the casein plastein was observed (Table
6). Casein plastein was heated in the presence of tyrosine
and the incorporation of tyrosine appears roughly equal to
that obtained with the ethyl ester preparation. Free
tyrosine was added to a casein peptic hydrolyzate concen—
trate and heated for 5 min at 100 C. The product was
washed with ethanol as above and amino acid analysis was
performed on.the pellet. The free tyrosine was insoluble
in the ethanol and was present in the pellet fraction,
amounting to 90% tyrosine.

The incorporation of L-tyrosine into soy hydroly-
zates gave similar results for both heated and enzymati-
cally activated plasteins. Soy plastein exhibited a 9.5-
fold increase in L-tyrosine content whereas the heated soy
hydrolyzate contained less than a 2—fold increase in tyro—
sine. Again free tyrosine showed a 7-fold increase over

the native soy protein (Table 7).

l

49

Table 6.--L-tyrosine ethyl ester incorporation into casein
p1astein.*

 

 

 

Samples T _

Amino _ Casein Casein

acid (acid TEE** Casein C3581? Hydroly- Hydroly—

hydrolysis) plastein Plastein zate + zate +

+ TEE Heat + Heat +

TEE Tyr051ne
Ala —- 2.73 1.35 2.78 0.20
Arg —- 3.90 1.11 1.92 0.27
Asp -- 7.14 4.25 9.32 0.66
Cys/2 -— 0.00 0.69 1.20 0.00
Glu -- 22.50 5.97 10.95 1.40
Gly -— 1.70 1.55 3.75 0.16
His —- 3.00 0.70 1.39 0.21
Ile -- 5.21 3.13 6.06 0.49
Leu -— 5.58 3.80 8.00 0.74
Lys —- 8.26 2.19 4.01 0.76
Met -- 0.80 0.56 1.08 0.00
Phe -- 5.29 2.96 5.20 2.15
Pro —— 11.79 4.23 7.45 0.60
Ser —- 5.54 3.75 7.41 0.51
Thr -- 4.10 2.51 5.36 0.52
Trp*** __ -_ __ -_ __
Tyr 10.0 5.79 58.36 18.69 90.94
Val -- 6.64 2.90 5.61 0.38

 

3FGram residues/100 grams protein.
**Gram free tyrosine/100 gram L-tyrosine ethyl ester.
***Not determined.

50

Table 7.-—L-tyrosine ethyl ester incorporation into soy
p1astein.*

 

 

 

 

 

Sample: 3
acigmiggid TEE** Soy SOY . Hydggly- Hydigly-
hydrolysis) plastein plastein zate + zate +

+ TEE Heat + Heat +
TEE Tyrosine

Ala -- 3.93 2.05 3.38 2.91
Arg -- 7.98 2.94 6.05 4.40
Asp -- 10.30 6.39 11.78 6.23
Cys/2 -- 0.73 0.63 0.53 0.48
Glu -— 13.76 8.13 14.17 7.64
Gly -— 3.38 2.17 3.67 2.74
His -- 2.13 0.93 1.56 1.52
Ile -- 7.07 3.25 5.73 4.70
Leu -- 11.02 4.97 8.64 7.56
Lys —- 6.54 2.50 4.11 3.36
Met -- 0.33 0.26 0.61 0.35
Phe -- 8.11 4.43 6.38 5.51
Pro -- 4.48 3.05 5.11 6.14
Ser -- 5.29 3.82 6.91 3.95
Thr -- 3.88 2.65 4.35 3.21
Trp*** __ -- __ _- _-

Tyr 10.0 4.77 49.21 12.09 35.17
Val —- 6.30 2.90 4.93 4.13

 

*Gram residues/100 grams protein.
**Gram free tyrosine/100 gram L-tyrosine ethyl ester.
***Not determined.

 

..:

.o

L.

n»

51

Horowitz and Haurowitz (1959) established that
ethyl ester of Cl4-labeled phenylalanine, tyrosine,
threonine, asparatic acid, glutamin acid, leucine, iso-
leucine, and histidine were incorporated into plastein.
They subjected their test specimens to extensive dialysis
at different pH values. The amino acid-incorporated plas—
teins were extracted with boiling water, acetone, and
dinitrofluorobenzene without significant loss of the
incorporated residues. Paper chromatography of these
plasteins showed no evidence of free phenylalanine or its
ethyl ester. A50 33 31. (1974) reported that L-lysine
could be incorporated by the same mechanism contradicting
the observations of Horowitz and Haurowitz (1959). The
lysine enriched plastein was washed with 10 volumes of 50%
ethanol made to 0.1 N with NaOH followed by 90 volumes of
diluted HCl and collected by centrifugation. Similarly
Arai 33 31. (1975a) reported that the methionine content
of protein could be increased through the incorporation of

the methionine ethyl ester.

CONCLUSIONS

The objective of this study was to investigate
various aspects of the plastein reaction. Chymotrypsin
appeared to be enzymatically active during the plastein
reaction and was responsible for the changes in physical
properties observed. There was no evidence that cycli-
zation, transamination, or transpeptidation reactions
occurred during the plastein reaction. Incorporation of
amino acids from their ethyl esters does not appear to be
the result of peptide bond formation. Instead, as the
apolar amino acids are cleaved from their ethyl esters
by Chymotrypsin, they aggregate and precipitate.

Hydrophobic interactions in proteins is complex
and seems to result from apolar amino acid side chains in
contact with a polar solvent (water). The water seeks to
maintain its structural integrity despite the interruption
of the polar amino acids. Thus, water becomes highly
ordered near aliphatic and aromatic apolar amino acid
residues. This entropy difference is the basis for the
hydrophobic effect.

The experimental evidence to support hydrophobic

interaction in plastein formation is considerable. Column

52

53

chromatography and disc gel electrophoresis results
indicated that there was no increase in the molecular
weight profile of the hydrolyzate attributed to the plas—
tein reaction. The heat-induced insolubility of peptic
digests was completely reversed by SDS. The specific
activity of pepsin exposes N-terminal phenylalanyl groups.
Chymotrypsin exposes C-terminal phenylalanyl, tyrosyl,
and tryptophanyl groups. These three amino acid residues
represent three of the four most hydrophobic amino acids

(Tanford, 1962).

 

LITERATURE CITED

LITERATURE CITED

Arai, S., Yamashita, M., and Fujimaki, M. 1973. Glutamyl
oligopeptides as factors responsible for tastes of
a proteinase-modified soybean protein. Agr. Biol.
Chem. Japan 33: 1253.

Arai, S., Yamashita, M., and Fujimaki, M. 1975a. A
parameter related to the plastein formation. J.
Food Sci. 33: 342.

Arai, S., Yamashita, M., and Fujimaki, M. 1975b. Plastein
reaction and its application. Cereal Food World
33(2): 107.

A30, K., Yamashita, M., Arai, S., and Fujimaki, M. 1973.
General properties of a plastein synthesized from
a soybean protein hydrolyzate. Agr. Biol. Chem.
Japan 31(11): 2505.

A30, K., Yamashita, M., Arai, S., and Fujimaki, M. 1974.
Tryptophan-, threonine—, and lysine—enriched
plasteins from zein. Agr. Biol. Chem. Japan
33(3): 679.

Bigelow, C. C. 1967. On the average hydrophobicity of
proteins and the relation between it and protein
structure. J. Theoret. Biol. 13: 187.

Boyer, P. D., ed. 1971. The Enzymes, 3rd ed. P. 156,
Academic Press, New York.

 

Brenner, H. D., and Niederwiesee, P. R. 1967. Methods in
Enzymology (S. P. Calowick and N. 0. Kaplan, eds.),
Academic Press, New York.

 

Catsimpoolas, N., Campbell, T. G., and Meyer, E. W. 1968.
Immunochemical study of changes in reserve protein
germinating soybean seeds. Plant Physiol. 33: 799.

Clark, J. M., Jr. 1964. Egperimental Biochemistry, lst
ed. P. 69, W. H. Freemand 8 Company, San Francisco.

54

55

Determann, H., and Wieland, T. 1961. Ein synthetisches
pentapeptid als plastein-monomers. Makromol.
Chem. 33: 312.

Determann, H., Sipp, O., and Wieland, T. 1962. Synthesen
Weitherer plastein-sktiver pentapeptide. Justus
Liebigs Annaler Der Chemic. 651: 172.

Determann, H., Bonhard, K., Kohler, R., and Wieland, T.
1963. Untersuchungen uber du plastein-reaktion
VI. Helv. Chim. Acta. 33; 2498.

Fujimaki, M., Yamashita, M., Arai, S., and Kato, H. 1970.
Enzymatic modifications of proteins in foodstuffs
Part I. Enzymatic proteolysis and plastein syn-
thesis application for preparing bland protein-
1ike substances. Agr. Biol. Chem. Japan 33(9):
1325.

Fujimaki, M., Kato, H., Arai, S., and Yamashita, M. 1971.
Application of microbial proteases to soybean and
other materials to improve acceptability, especially
through the formation of plastein. J. Appl.
Bacteriol. 33(1): 119.

Fujimaki, M., Utaka, K., Yamashita, M., and Arai, S. 1973.
Production of higher-quality plastein from a crude
singlecell protein. Afr. Biol. Chem. Japan 31(10):
2303.

Gordon, W. G., Sennett, W. F., Gable, R. S., and Morris, M.
1949. Amino acid composition of a—casein and B-
casein. J. Am. Chem. Soc. 11: 3293.

Horowitz, J., and Haurowitz, F. 1959. Mechanism of
plastein formation. Biochim. Biophy. Acta. 33:
231. _H'

Lehninger, A. L. 1971. Biochemistry, 3rd ed. P. 170,
Warth Publishers Inc., New York.

 

Mangino, M. E. 1973. M.S. Thesis, Michigan State Univer-
sity.

Moore, 8., and Stein, W. H. 1954. Procedures for the
chromatographic determination of amino acids on
four percent cross-linked sulfonated polystyrene
resins. J. Biol. Chem. 311: 893-906.

Moore, 8., Spackman, D. H., and Stein, W. H. 1958.
Chromatography of amino acids on sulfonated poly-
styrene resins. Anal. Chem. 33: 1185.

56

Neurath, H. 1963. The Proteins Vol. 1. P. 477, Academic
Press, New York.

 

Rakis, J. J. 1961. Amino acids in soybean hulls and oil
meal fractions. J. Agr. Food Chem. 3: 409.

Reynolds, J. A., and Tanford, C. 1970. The gross con-
formation of protein—sodium dodecyl sulfate com-
plexes. J. Biol. Chem. 33(19): 5161.

Rick, W. 1963. Methods in Enzymatic Ana1ysis (H. D.
Bergmeyer, ed.). Academic Press, New York.

 

Spackman, S. H., Stein, W. H., and Moore, S. 1958. Auto-
matic recording apparatus for use in the chroma-
tography of amino acids. Anal. Chem. 33: 1190-1206.

Smith, A. K., and Circle, S. J. 1972. Soybeans: Chemistry
and Technology, Vol. 1. P. 128, Avi Publishing
Co., Westport, Connecticut.

 

 

Subbaiah, P., and Thompson, G. 1974. Studies of membrane
formation in Tetrohymena pyriformis. J. Biol.
Chem. 249(4): 1302.

 

Swank, R. T., and Munkres, K. D. 1971. Molecular weight
analysis of oligopeptides by electrophoresis in
polyacrylamide gel with sodium dodecyl sulfate.
Anal. Biochem. 33: 462.

Talbot, D., and thantis, D. 1971. Fluorescent monitoring
of SDS gel electrOphoresis. Anal. Biochem. 44:
246. _—

Taminato, S., Yamashita, M., Arai, S., and Fujimaki, M.
1972. Probes for catalytic action of a-
chymotrypsin in plastein synthesis. Agr. Biol.
Chem. Japan 33(9): 1575.

Tanford, C. 1962. Contribution of hydrophobic inter-
actions to the stability of the globular confor-
mation of proteins. J. Am. Chem. Soc. 33: 4240.

Tasi, S., Yamashita, M., Arai, S., and Fujimaki, M. 1972.
Effect of substrate concentration of plastein pro-
ductivity and some rheological properties of the
products. Agr. Biol. Chem. Japan 33(6): 1054.

Tasi, S., Yamashita, M., Arai, S., and Fujimaki, M. 1974.
Polyacrylamide gel electrophoresis of plasteins.
Agr. Biol. Chem. Japan 33(3): 641.

57

Tauber, H. 1951a. Synthesis of protein-like substances
by Chymotrypsin. J. Am. Chem. Soc. 13: 1288.

Tauber, H. 1951b. Synthesis of protein-like substances
by Chymotrypsin from dilute peptic digests and
their electrophoretic patterns. J. Am. Chem. Soc.
73: 4965.

Virtamen, A. 1., and Kerkkonen, H. K. 1948. Structure
of plasteins. Nature 161: 888.

Wasteneys, H., and Borsook, H. 1930. The enzymatic
synthesis of protein. Physiol. Rev. 13: 110.

Weber, K., and Osborn, M. 1969. The reliability of
molecular weight determination by dodecyl sulfate
polyacrylamide gel electrophoresis. J. Biol.
Chem. 333: 4406.

Whitaker, J. R. 1972. Principles of Enzymology for the

Food Sciences. P. 538, Marcel Dekker, Inc., New
York.

 

 

Yamashita, M., Arai, S., Matsuyama, J., Gonda, M., Kato,
H., and Fujimaki, M. 1970. Enzymatic modifica-
tions of proteins in foodstuffs Part III. Phenom—
enal survey on a-chymotrypsin plastein synthesis
from peptic hydrolyzate of soy protein. Agr.
Biol. Chem. Japan 33(10): 1484.

Yamashita, M., Tasi, S., Arai, S., Kato, H., and Fujimaki,
M. 1971a. Enzymatic modification of proteins
in foodstuffs Part V. Plastein yields and their
pH dependence. Agr. Biol. Chem. Japan 33(1): 86.

Yamashita, M., Arai, S., Tasi, S., and Fujimaki, M. 1971b.
Plastein reaction as a method for enhancing the
sulfur-containing amino acid level of soybean
protein. J. Agr. Food Chem. 13(6): 1151.

Yamashita, M., Arai, S., Tanimoto, S., and Fujimaki, M.
1973. Condensation reaction occurring during
plastein formation by a-chymotrypsin. Agr. Biol.
Chem. Japan 31(4): 953.

Yamashita, M., Arai, S., Kokubo, S., Aso, S., and Fujimaki,
M. 1974. Plastein with an extremely high amount
of glutamic acid. Agr. Biol. Chem. Japan 33(6):
1269.

APPENDIX

APPENDIX

Enzyme Activity

 

Tris-Buffer

 

A 0.05 M tris buffer was prepared by dissolving
6.057 g tris-hydroxymethyl aminomethane in 900 m1 of
distilled water and adding 23 m1 of 2N HCl. This solution

was diluted to 1 1t after the pH was adjusted to 7.0.

Column Chromatography

 

Column Buffer

 

A 0.1 M tris—HCl buffer was prepared by adding
12.114 g tris—hydroxymethyl aminomethane to 900 m1 dis-
tilled water. 58.5 g sodium chloride was added and suf—
ficient 2N HCl was added to reach pH 8.0. This solution

was diluted to 1 1t.

Sample Buffer

 

0.5 ml of a 1/2% protein solution was added to
1/2 ml of a 0.01 M phosphate solution containing 1% SDS

and 0.02% sodium azide.

58

59

Polyacrylamide Gel Electrophoresis

 

TEMED

 

N,N,N',N',-Tetramethylethylenediamine

Staining Solution

 

To 454 m1 of 50% aqueous methanol, combine 1.25 g

Comassie Brilliant Blue, and 46 ml glacial acetic acid.

Destaining Solution

 

75 m1 of glacial acetic acid is combined with 250

ml methanol, and 675 m1 of distilled water.

Dansyl Chloride

 

1-dimethy1amino-5-naphtha1enesu1fony1 chloride was
made 10% (100 mg/ml) in acetone and packed under nitrogen.
Dansyl chloride was purchased from the Pierce Chemical

Company.

SDS Phosphate Gel Buffer

 

7.8 g of NaH PO - H O was combined with 38.6 g of

2 4 2

NaZHPO4 - 7H20 in 900 ml of distilled water. 2 g of SDS
was added and 0.02% sodium azide was added to prevent
microbiological growth. The solution was then diluted to

1 1t.

SDS Phosphate Running Buffer

 

SDS phosphate gel buffer was diluted 1:3 with dis-

tilled water.

60

Tris Glycine Running Buffer

 

0.046 M tris glycine buffer was prepared by adding
5.6 g tris to 28.8 g glycine and adjusting the pH to 8.3
with glycine. 2 g SDS was added and using distilled water

the solution was diluted to 1 1t.

Tris Glycine Spacer Buffer

 

0.062 M tris buffer was prepared by adding 7.5 g
tris 50 800 ml distilled water and adjusting the pH to
6.7 with 2 N HCl. This solution was made 2% in SDS and

diluted to one liter.

Tris Glycine Gel Buffer

 

0.76 M tris was made 0.1% with SDS and adjusted to

pH 8.9 with glycine.

Ninhydrin

 

Ninhydrin Solution

 

400 mg of stannous chloride dihydrate was dissolved
in 250 m1 of 0.2 M acetate buffer at pH 5.0. This solution
was mixed with 250 ml of methyl cellosolve (ethylene
glycol monomethyl ether) containing 10 g of dissolved nin-

hydrin and stored in a glass bottle at 0 C.

Citrate Buffer

 

4.3 g citric acid was combined with 8.7 g Na3

Citrate ° 2H20 in 250 m1 of solution. This solution was

adjusted to pH 5.0 with NaOH or HCl.

61

 

Kjeldahl
Digestion Mixture
5.0 g of CuSO4 ° SHZO and 5.0 g of SeO was made up
to 500 ml with concentrated H2804.

Indicator Solution

 

400 mg bromocresol green and 40 mg ethyl red were

dissolved in 100 ml of 95% ethanol.

Thin Layer Chromatography

 

Ninhydrin Spr3y

 

0.039 g of ninhydrin was dissolved in 350 m1
absolute ethanol, 14 ml colodine, and 135 m1 glacial

acetic acid. This solution was stored at 0 C.

 

Pepsin hydrolyzed sample - protein .

62

% Hydrolysis Calculation

 

 

Acid hydrolyzed sample — protein

Amino

acid

Try
Ile
Tyr
Phe
Pro
Leu
Val
Lys
Met
Cys/2
Ala
Arg
Thr
Gly

100 = % Hydrolysis

Hydrophobic Calculations

 

H¢

(kcal/residue)

3.00 Each amino acid is (m
2.95

2.85 multiplied by the HO value.
2.65

2.60 This product is totaled for
2.40

1.70 all the amino acids and
1.50

1.30 divided by the number of 13
1.00

0.75 amino acids in the protein.

0.75

0.45 This is the average hydro-

0.00

 

phobicity of a protein and is

expressed in cal/residue.

 

ImIII111111mum11111111111I

307