[Agr.

Biol.

Chem.,

Vol.

30,

No.

7,

Carbohydrate

p.

646•`650,

1966]

Component Casein

in 7S Protein

of Soybean

Fraction*

By Ikunori KOSHIYAMA NodaInstitutefor Scientific Research,Noda-shi,Chiba-ken, Japan ReceivedJanuary 25, 1966 Carbohydratecomponentsin the 7S protein from soybeancasein fraction were found to be mannase and hexosamine. The former was identifiedby paper and starch-column chromatographiesand its contentwas approximately4%, per protein. The latter, hexo samine,was containedabout 1.2%per protein. Mannosewas consideredas an integral constituentof the 7S protein from the data of heat and acid denaturation, paper electrophoresisand column chromatographywith SephadexG-200. INTRODUCTION

MATERIALS

It has been reported that soybean protein, 11S and 7S components of which account for about 70%, contains carbohydrate.2) Recent ly, Roberts and Briggs2) found that there was large difference in carbohydrate content be tween 7S and 11S component. By the way, they had prepared 11S protein (80% in purity) from the cold-insoluble fraction and purified 7S protein (90% in purity) by a process in volving ammonium sulfate fractionation and gel filtration with Sephadex G-100. The content of carbohydrate has been considered to be a large difference in the properties of the both components. But, it has not been proved whether carbohydrate component is the integral constituent or the contaminated substance in 11S and 7S proteins. The present paper deals with the carbo hydrate component in an ultracentrifugally and electrophoretically pure 7S protein of soybean casein fraction.3) *

A part

of

this

report

was

presented

at

the

general

meeting of the Kanto Branch of the Agricultural Chemical Society of Japan, Kofu, October, 16, 1965. 1) J. Tillmans and K. Philippi, Biochem. Z., 215, 36 (1929). 2) R. C. Roberts and D. R. Briggs, Cereal Chem., 42, 71 (1965). 3) 1. Koshiyama, This journal, 29, 885 (1965).

AND

METHODS

7S Protein Sample. The purified 7S protein

was prepared

from

casein

method

the

fraction

by

the

of

soybean previous

papers.s,4) 11S Protein

Sample.

The 11S protein was prepared from the cold-insoluble fraction of water-extractable soybean protein and purified by the method of Mitsuda et al.5) Ultracentrifugal analysis. Sedimentation analysis was carried out at room temperature with a Hitachi UCA-1 ultracentrifuge at 55430 r.p.m. The solvent was the same buffer as that used on column chromatography with Sephadex G-200. Determination

of total

carbohydrate.

Phenol-sulfuric acid method according to Dubois et al.6) was used for determination of total carbo hydrate. Paper

electrophoresis.

Paper electrophoresis was performed on 15 x 40 cm filter paper for fifteen hours with 150v-15 mA, using 0.1M phosphate buffer of pH 7.60 as the buffer. The zone of Ponceau 3R.

protein

4) I. Koshiyama (1965). 5) H. Mitsuda, T. 29, 7 (1965). 6) M. Dubois, K. and F. Smith. Anal.

and

was

N.

Kusano

detected

Iguchi,

This

by staining

Journal,

and K. Hasegawa,

A. Gilles, J. K. Hamilton, Chem., 28, 350 (1956).

This

with

29,

144

Journal,

P . A. Rebers

Carbohydrate

Determination

of

Protein

protein

content

at

660

mƒÊ,

at

280

mƒÊ

Column

was at a

mƒÊ P-1

chromatography

formed

at

The

eluant

7.60,

0.5

2•`4•Ž used

toethanol

and

was

0.01M

previous

buffer,

0.01M

fifteen

water,

pH

1ml

fraction

from

7S

cell

at

sample

The tion

from et

protein

al.9)

follows:

hours.

After

with

the

10%

was

and

The

effluent,

up

in

was

of

next

sugars

colour

a spectrophotometer

using

content.

was

determined

water

with

by

standard

Elson

(1 x

the

and

to

pre

curve.

this

AND

of

DISCUSSION

carbohydrate.

experiment,

purified

TABLE

15 cm)

7S

as protein

water,

I.

shown from

CARBOHYDRATE

PURIFIED

meshes.

of

used

in

Table

soybean

I, casein

1•`2ml

100•`200

carbohydrate,

was

times.

in

100ml

In the

three

column

a

Identification

neutralized

dissolved

of

pare

Glucosamine

RESULTS

two

centrifuged,

the

contained

for

7S AND

CONTENT lIS

IN THE

PROTEINS

and

was

dried

three

in

the

solvents

7S

1)

Phenol:

2)

Pyridine.

3)

n-Propanol:

were

used

for

detection

of

protein.

water=75:25

(by

ethyl

The

filter

20•Ž

with

by

acetate:water=

paper

(Toyo

aniline

7:1:2

(by

out

Gardell's

was

developed and

stained

reagent.10)

chromatography using

Hanafusa

fraction

paper)

chromatography.

Starch-column

each

50

chromatography

hydrogen-phthalate

Starch-column

carried

No.

ascending-type

et

effluent

the

method

al.9)

The

solution

collector method.12)

was

of

carbohydrate

described amount

collected determined The

1)

measured

by

Lowry's

2)

measured

by

orcinol-sulfuric

method

and acid

turbidity method

at at

420

420mƒÊ. mƒÊ.

volume).

volume).

in

in

The

chromatography.

The

and

bath

immersion

vacuo.

Paper

at

heated

water

added.

hexosamine

content method.13)

was

100•Ž was

(H-form)

washed

which

was

were

(400mg) at

hot up, to

CG-120

column

the

with

transferred

Amberlite

protein

and

dried

by

procedure

hydrolyzate

washed

(1ml)

in with

mƒÊ.

Determination

Morgan's

frac

described

the

acid

hydroxide

was

water

7S

acid

supernatant

with

method of

sulfuric

barium

precipitate The

2N

carbohydrate

the

details

purified

with

100ml

a boiling

was by

to

by

of

acid

analyses. of

was

The

The

hydrolyzed

of

carbohydrate procedure

7S

Hanafusa as

for

preparative

in cooling

ethanol

measured

Hexosamine protein

solution

by

50ml

trichloroacetic

solution After

prepared of

diluting

effluent

370

was

mixture

8.5N

and

reagent

of

were

1cm

the

of

minutes.

densities

2-mercap

chlorides)

carbohydrate

The of

647

solution to

stirring

1ml

for

reagent

32ml

bath,

Fraction

aniline

and

ice

with

per

papers)

phosphate

sodium

of

the

of

n-butanol.

G-200. was

Casein

The

2ml

n-butanol an

G-200

to

follows:

adding

absorption

Sephadex

containing

0.4M

Preparation

the

Sephadex

strength,

method

spectrophotometer.

according

ionic

Lowry's7)

and

with with

of Soybean

as by

420

Hitachi

Chromatography

in 7S Protein

content.

measured

turbidity with

Component

procedure

by of

with

was Gardell11)

carbohydrate an

by

automatic a

was

modified modified

\ 7) O. H. Lowry, N. J. Rosebrough, A. L. Farr and R. J. Randall, J. Biol. Chem., 193, 265 (1951). 8) K. Hasegawa, T. Kusano and H. Mitsuda, This Journal, 27, 878 (1963) 9) H. Hanafusa, T. Ikenaka and S. Akabori, J. Biochem., 42, 55 (1955). 10) S.M. Partridge, Nature, 164, 443 (1949). 11) S. Gardell, Acta Chem. Scand., 7, 201 (1953). 12) S. Gardell, ibid., 5, 1011 (1951).

fraction contained approximately 5% carbo hydrate per protein as glucose. This per centage of carbohydrate content by phenol sulfuric acid method corresponded well with the value by orcinol-sulfuric acid method.14) Bial's reaction15,16) to the 7S pro tein solution for pentose was negative. As shown in Fig. 1, the ultracentrifugally purified 11S protein which had S20,w value of 11.62S, contained about 0.8% of carbo hydrate per protein as glucose. So, there was a clear difference in carbohydrate content between 11S and 7S protein. For the purpose of confirming whether the 13) L.A. Elson and W. T. Morgan, Biochem. 1., 27, 1824 (1933). 14) W. R. Fernell and H. K. King, Analyst. 78, 80 (1953). 15) A. H. Brown, Arch. Biochem., 11, 269 (1946). 16) W. Mejbaum, Z. Physiol. Chem., 258, 117 (1939).

648

Ikunori

KOSHIYAMA

TABLE II. CARBOHYDRATE CONTENT AFTER HEAT DENATURATION OF THE PURIFIED 7S PROTEIN

*

measured

and

heated

After

Photographs were taken at 55430r.p.m. Direction to left.

after 45min. of of sedimentation

to

or

in

7S

contaminated

experiments Reducing

On

heating

protein

with

was

in were

1)

protein

3)

centrifugation is from right

the

in

protein,

the

the

next

several

by

solution solution,

of no

centrifugation

reducing

not

The

7S

in

of

one

reducing

sugar

sugar

hydrochloric

7S

sugar

carbohydrate

the

7S

probably

in

glucosides. to

and,

by

the

with

the

of

contain

had

no

minutes.

centrifuged.

As

carbohydrate

shown

reaction

acid was of protein.

intensively

with given

to

denaturation. was

trichloroacetic solution. precipitate

the

done

acid As

by to

shown gave

adding

1ml

in strongly

1ml

of Table

0.5%

III, positive

reaction.

TABLE III. CARBOHYDRATE CONTENT AFTER ACID PRECIPITATION OF THE PURIFIED 7S PROTEIN

the

most

seemed

of

N-glucosides part

measured

by

phenol-sulfuric

acid

method

at

490

re

of in

of

the

PAPER

IV.

CARBOHYDRATE

AFTER

PAPER

DISTRIBUTION

ON THE

ELECTROPHORESIS

OF

7S PROTEIN

of exist

or

that

TABLE

re

part to

mƒÊ .

4) Paper electrophoresis. A portion of 0.25ml of 1 % protein in aqueous solution was subjected to electro phoresis. After electrophoresis, both edges

with

amount

protein

protein

at 0.7%

hydrolysis

small

small

the

method.

mild

form the

protein,

protein

obtained,

But,

combine

7S

only

the

7S

acid

to

Somogyi's

the

in

ten

mƒÊ .

protein

25mg

hydrochloric

by

was

the

proved

that

acid,

ducing

or

490

method.""

heating

0.5N hour,

Considering ducing

by

with

for

at

filtration.

the

Somogyi's of

obtained

protein

100•Ž

by

hydrolyzate

was

20%

five

method

precipitate

and

sugars

determined acid

which

for

was

precipitation

Molisch's

*

were

100•Ž it

II,

Acid

Acid

protein only

combined

7S protein.

aqueous

Fehling's

course,

was

Table

acid

examined.

sugar

detected

Of

the

to

phenol-sulfuric the precipitate

of carbohydrate

phenol-sulfuric

cooling,

in

FIG. 1. Ultracentrifugal Patterns of the Cold insoluble Fraction (upper) and the Purified 11S Protein (lower).

by

0-

seemed form

of

polysaccharides. 2)

Heat

One to

0.5ml

ml

denaturation. of of

0.5% 1M

protein

solution

phosphate

buffer

17) J. Nikuni, "Starch p. 686 (in Japanese).

Chemistry"

Asakura

was of

added pH

Shoten

6.0 (1957), *

measured

by

phenol-sulfuric

acid

method

at

490 mƒÊ.

THE

Carbohydrate

Component

in 7S Protein

of the filter paper were cut out and the zone of protein was stained. On the remaining filter paper, four separate parts of A, B, C, and C2, 2cm wide each, were cut as shown in Table IV, and eluted with water, and the quantities of carbohydrate in each fraction were determined. As shown in Table IV.

of Soybean

Casein

Fraction

649

an intensive colour reaction of carbohydrate was given to the stained part of protein with Ponceau 3R. 5) Column chromatography with Sephadex G200. As shown in Fig. 2, the intensities of carbo hydrate reaction of each fraction ran parallel to the protein contents. There had been a question18) whether 7S protein was a glycoprotein or not. However, considering the results from 1) to 5), it could be concluded that the carbohydrate in the 7S protein combined tightly with the protein and made a protein-carbohydrate complex as a glycoprotein. Qualitative and quantitative analyses of carbo hydrate. The nature of the carbohydrate in the 7S

FIG. 2.

Column

Protein

with

Chromatography Sephadex

of the Purified

G-200

7S

(2 x 200cm).

Sample: 100mg of the purified 7S protein in 5ml of the eluting buffer. Eluant: 0.01M phosphate buffer, pH 7.60, 0.5 ionic strength, containing 0.01M 2-mercaptoethanol and 0.4M sodium chloride. Flow rate: about 10ml/hr. Fraction volume: 5ml.

FIG. 3.

Paper A: B: C:

protein was determined by paper column chromatographies. 1) Paper chromatography. The fraction of carbohydrate

in the

7S

protein, separated from amino acids and peptides, was subjected to one dimensional chromatography on filter paper. As shown in Fig. 3, the RF value of the sugar in the 7S protein coincided with that of mannose,

Chromatograms of Carbohydrates in the Purified and of Standard Carbohydrates. Phenol:water=75:25 Pyridine. n-Propanol:ethyl

and starch-

7S Protein

(by volume). acetate:water=7:1:2

(by volume).

18) H. W. Schultz and A. F. Anglemier, "Symposium on Foods, Proteins and Their Reactions" The Avi Publishing Co., Inc. (1964) p. 163.

650

Ikunori

KOSHIYAMA

of the 7S protein. For the standard solution of carbohydrate, mannose was used. The result is shown in Table V

FIG.

4.

Starch-column

Mixture tion

of

Column: =4:1:1

Chromatography

of Monosaccharides the

Purified

1•~20cm. (by volume).

and

of a Synthetic Carbohydrate

Frac

TABLE V.

SUMMARY

7S Protein. Solvent: Fraction

n-butanol: volume:

n-propanol:water 1 .17ml.

using three different solvents. Any spot of carbohydrates except mannose could not be detected on the paper. 2) Starch-column chromatography. Diagrams in Fig. 4 represent the results obtained with a synthetic mixture of mono saccharides and with the carbohydrate frac tion of the 7S protein. From these experi ments, it was considered that carbohydrate in the 7S protein consisted of mannose. The phenol-sulfuric quantitative

3) Hexosamine content. The 7S protein showed positive hexosamine reaction as usual glycoprotein. Hexosamine content is shown in Table V Determination of the kind of hexosamine should be further investigated.

acid method determination

was used for the of carbohydrate

THE AMOUNTS OF TOTAL CARBOHYDRATE

AND HEXOSAMINE IN THE PURIFIED

Contents of carbohydrate per protein, respectively.

and hexosamine

7S PROTEIN

were calculated

The purified 7S protein from soybean casein fraction contained approximately 4% mannose per protein. Mannose in the protein com bined tightly with the protein and seemed to make a protein-carbohydrate complex as a glycoprotein. Hexosamine which tained in the 7S protein was about

was con 1.2% per

protein. Acknowledgement. The author wishes to express his sincerest thanks to Prof. Y. Sakurai and Assoc. Prof. M. Fujimaki of the Uni versity of Tokyo for their kind guidance and encouragement throughout this work. Great indebtedness is acknowledged to Prof. K. Arima and Prof. Y. Ikeda of the University of Tokyo for their valuable suggestions. Thanks are due to Department of Agricultural Chemistry, the University of Tokyo, for ultracentrifugal analysis. The author also wishes to thank Dr. M. Mogi and Dr. N. Igu chi for their encouragement and Miss T. Ichi kawa for her kind assistance.