N-terminal group of ovalbumin

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATtONS

N-TERNINAL

CROUP OF OVALBUMIN

Vol.& No.2,1961

Kozo Narita Institute for Protein Research, Osaka University, Osaka, Japan

Received Ma.y 15, 1961 Despite group

many efforts

of ovalbumin,

by the

usual

C-terminal 1955).

no conclusive

N-terminal proline

Thus

several

are a loop

peptide

Ottesen,

1956;

group.

No positive

a carbohydrate

moiety

1956).

The possibility

by the

isolation 1954).

linked

oligosaccharide

different --et al, isolated

In

Jevons,

glycopeptides

1938),

1958). from

Anfinsen

the

and Redfield, was excluded

in which group

peptides the

with

constituent

has been

The present

'160

with

of phosphoserine

(Cunningham

the

structure,

one was N-phosphoryl-

p-carboxyl of all

terminal

was N-substitution

1950;

aspartic

These

1952;

latter

a glycopeptide

groups

detection

former.posoibility

the

two kinds

turn

the

reason

suggested.

of N-phosphorylation

consisting

research

been

As to the

(Porter,

(Neuberger,

1958;

the

and the other

through

and mannose

for far.

of the

the

and an N-substituted

were considered: 1954)

part

1960)

thus

(Flavin,

(Flavin,

have

a

and Fraenkel-Conrat,

(Linderstrom-Lang,

results

two possibilities

(Niu

although

has escaped

analysis

Haruna,

has been obtained

to explain

structure

have been reported

ation

found

possibilities

group

N-terminal

methods,

of ovalbumin

terminal

the

result

analysis

has been

why the N-terminus of the

to find

by three

1957;

author

10% trichloroacetic

an glucosamine

isolated

al -et -9

peptidic

Johansen

has also acid-

Vol.5,No.2,19iSl

BIOCHEMICAL

soluble

peptic

sieve

digest

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of ovalbumin

of Dowex 50 columns

peptidea

could

column the

fraction

carbohydrate

content

fractionated at pH 5.6.

Two main

isolated. might

Thus the

the

1958)

hours. X2

(2.0

column

washed with

x

water

until

and washings

alkaline

The acidic

column,

and which

phoaphoserine

peptidea

by the

200~400 peak using figure

chloride

mesh)

in

zone electrophoreais

were homogeneous

with

protein

group ruled an acyl

out. group

of tobacco

mosaic

proteins. ovalbumin

the

proteinaae

et al,

1959)

with

an acid mesh)

the washings hydrolysis

peptide

were

cysteic

of Dowex 2-X8

shown in Fig.

acid-water

(4:l:l). peak

161

24

of Dowex 50column

was

1956).

The and

passed

through

acid

peptides,

was further

fractio-

column

(1.5

1. Homogeneity paper

for

collected

contain peptidea

by

al -et -9 which

and N-acyl

produced

ninhydrin-

fraction,

supposedly

was

form

became (Hira

(1 g.)

at pH 8.0

and the

100 ml.)

except

were

was entirely

by one dimensional

n-butanol-acetic

were further

the N-terminal

the

50-,100

(about

reaction

glycopeptidea

that

with

form

as is

was examined

block

acid-oxidized

25 cm.,

effluent

nated

anthrone

glycopeptidea

was treated

after

the

by the

(Nomoto

negative

lyophilized.

in

digestion

griaeua

The digest

was 38.4s

and other

The performic

Streptomscea

fractionated

of N-substitution

virus

to the

thus

by carbohydrate

as was found

subjected

of Dowex 50-X4 of

possibility

was considered

The glyco-

The yield

powder

possibility

(Narita,

form

ninhydrin-positive

be substituted

Finally

1958).

a X8 column.

The crude

by cellulose

of molecular

from

measured

in weight.

aid

al -et -9

on an acid

through

glycopeptide

and 1.2$

(Narita

be retained


by the

x 23 cm.,

of

each

chromatography No peaks

3. The results

in

the

of amino

Vol. 5, No. 2,196l

BIOCHEMICAL

AND BIOPHYSICAL

40

0

80

RESEARCH COMMUNICATIONS

180 FI?WTKIN

I20

Figure 1. Chromatographic fractionation on a column of Dowex 2-X8 (chloride form, 1.5 x 23 cm.) of the acidic peptide fraction from the enzymatic digest of ovalbumin. Solid and dotted lines show the ninhydrin color values before and after alkaline hydrolysis respectively. One fraction was 5 ml. The break-through peak was large neutral peptide fraction.

acid

and N-terminal

Val.CyS03H

(Rf 0.20)

and Anfinsen

(1954).

positive

cysteic

as well

as several

analyses

indicated

which

was already

Peak

acid

iodide-starch-positive spots them

with

was predominant

equimolar

gram of the peptide

hydrazinolyzate

showed the

hydrazide ammoniacal

(Rf

silver

0.45,

hydrazide 0.11)

nitrate

which reagent. 162

peptides but

the

paper.

Rf 0.46)

chlorine-

(Rf

above

One of

and contained

and glycine.

of acetyl

9:1:4;

0.37;

from

of the

presence

serine

5:3:2),

ninhydrin-

The ninhydrin-negative

1 mg.,

of serine

pyridine-aniline-water, water,

water

(about

quantities

several

ninhydrin-negative

3 was

by Flavin

or phosphoserine

components.

were extracted

peak

isolated

1 contained

peptides

other

that

Paper

chromato-

ninhydrin-negative

hydrazide

(Rf

0.65,

collidine-picoline0.46;

0.16)

were revealed One could

and glycine with

not

the be certain

Vol. 5, No. 2,196l

which

BIOCHEMICAL

was the

C-terminal

results

of hydrazinolysis

glycine

and serine

by the

action

hours.

Therefore

gave rise

the

The results

was decided Confirmation

of the

synthesis

indicate

the

Neuberger acetylation tion isolated

from

1961) reported enzymatic

digest

the

for

several

was incubated

with

from

(Niu

peptide

group.

of the

group.

1955)

Consequently

acetyl

The present

results

of ovalbumin

Johansen,

the

it

the

ovalbumin

results

(Marshall

of an acetyl

of ovalbumin,

but

the

strongly to

and

of N-terminal

and in

they

peptide

appears

Marshall

presence

from

isolation

the

by the

present

group

Recently

of

had to be N-acetyl.Gly.Ser.

structure

in

analysis

and Fraenkel-Conrat,

terminal

protein

group it.

at 100"

partly

was liberated

suggested

of the

of acetyl

hydrazides

serine

by acetyl

(1960)

C-Terminal

chromatogram

the N-terminal

be substituted

above

The paper

is now in progress.

that

the

24 hours.

as the

that

to their

peptide

method

serine

from

alone.

of the C-terminal

hydrazinolysis-DNP

by

acetyl

RESEARCH COMMUNICATIONS

judging

hydrazine

showed that

gave also

acid

experiment

A for

incubate

peptide.

amino

of anhydrous

carboxypeptidase the

AND BIOPHYSICAL

of the

estima-

glycopeptide and Neuberger, peptide

structure

from was not

described. References c. B. and Redfield R. R. Advances in Protein Chem., 11, 1 (19563. B. J. and Nuenke, R., Biochim. Cunningham, Il. w., Nuenke, Biophys. Acts, 26, 660 (1957). Flavin, M., J. Biol. Chem., 210, 771 (1954). Flavin, M. and Anfinsen, C. B., J. Biol. Chem., 211, 375 (1954). Haruna, I., J. Biochem. (Japan), 42, 755 (1960). Hirs, C. H. W., Moore, S. and Stein, W, H., J. Biol. Chem., 219, 375 (1956). Jevons, F. R., Nature, 181, 1346 (1958). Johansen, P., Marshall, R. D. and Neuberger, A., Nature, 181, 1345 (1958). Anfinsen,

163

BIOCHEMICAL

Vol.5,No.2,1961

Johansen,

P.,

Marshall

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

R. D. and Neuberger,

J., 12, 239 tl960).

Linderstrom-Lan

(1952 7

, K.,

Stanford

Univ.

A.,

Publ.

Med.

Biochem. Sci.

No.

6

l

Marshall, Narita, Narita,

K., K.,

R. D. and Neuberger, A., Biochem. J., in press. Biochim. Biophys. Acta, 28, 184 (1958). Fujiwara, S. and Murasawa, S., Bull. Chem. Sot. Japan, 2, 381 (1958). Neuberger, A., Biochem. J., 2, 1435 (1938). Niu, C. I. and Fraenkel-Conrat, H., J. Am. Chem. Sot., Nomoto,

M and Narahashi,

Ottesen, Porter,

M., Arch. Biochem. R. R., Biochem. J.,

12, 5882 (1955).

(1959).

Y.,

J.

Biochem.

Biophys.

(Japan), a,

46, 473 f195oL

'164

46,

70 (1956).

1481