Sequence studies on bovine α-chymotrypsin

Vol.

21, No.

5, 1965

BIOCHEMICAL

SEQUENZE

AND

STUDIES

BIOPHYSICAL

ON 80’1

Stanley

N

C.

RESEARCH

COMMUNICATIONS

K-CHYU)TRYPSIN1

Glauser*

and Henry

Wagner3

Department

Temple

Received

November

The

Keil,

by the When

et al

(19631,

these

search 7,

acid

of

was

8 and

sults

of

undertaken

to

the

acid

analysis.

Paper

tank.

Eastman

1. This work 2. Established 3.

of

(2OO:l). Chromasheets

resolve

been

determined

discrepancy

this

4

- chymotrypsin

sequence by

Our is

This

noted work

the

13

(1958). re-

at

residues

confirms

the

amino

for,

Heedom

evident.

difference A.

The

A

acids

reat

the

A.

(Calbiochem

solvents

were

used were

Thin-layer was

A-grade,

was employed

chromatography Chromatograms

The

phenol-ammonia and

A chain

one

chymotrypsinogen

(1965).

chymotrypsinogen

&-chymotrypsin

identification.

ascending

of

is

bovine

et al

also

has

to

end

for

and Sorm,

there

attempt

chymotrypsinogen

Commercial

determined

(19641,

compared

amino The

of

been

O(-chymotrypsin are

the

has

Hartley

sequences

Hartley.

N terminus

sequence

oxidized

9 from

Pharmacology School of Medicine Pennsylvania

8, 1965

amino

A chain

of

University Philadelphia,

also

run

on

#53251)

as the primary 24.5

butanol-acetic

chromatography used.

lot

Chromatograms

x 24.5

cm

acid-water on silica-gel were

was used method paper

for

of amino in

a Shandon

(12:3:5) coated

developed

and plates

with

been supported by U.S. Public Health Service Grant AM 10072-01. Investigator of the American Heart Association and Associate Professor of Pharmacology, Temple University School of Medicine. Merck Sharp & Dohme Research Fellow. has

494

Vol.

21, No.

5, 1965

ninhydrin

dip

the

soluble

ether

reagent.

saturated

derivatives

were

amino-acid

analysis

a mcdification Primary

of four

ed

the

at up

in

1.0 dissolve,

soluble

Total

A-chain,

ing

very

with

senting

the

the purest

Leucine

hours,

after

which the it the

was

15

addition was

hours of

number

of

assumed

taken

that

amino-acid results

at

at

120

A-chain

and

the

addition

the

end

of

Giving serine

The

first

and

used

residue

this, the = 0.9,

three

another liberated

time

analyzed

amount

of

leucine

495

pg

to

eluted

220

mk. to

w

indicat-

analysis. with the

leucine

20

kg

after

after

140

glycine

observed

=

and

valine

hours,

amino-acid,

hours on

24

Since

more

24

DIFP-

liberated

C-terminal for

be

repre-

glycine

quantitatively

1.85,

borate

contamination

of

one

the

40°C

The

carboxypeptidase.

only to

280

sequence

of

fail-

fractions,

digestion,

20

heated

at

made

amount

penultimate was

peak

digested

analysis

which

at

this

the

material

A-chain

preparation

peak

was

of

the

this

, although

for

oxidized

absorbancy

absorption

C chains

had

medium

no

mg for

B-chain. and

their

hundred

material,

the

showed

increased of

carboxypeptidase a proline

by

8 hours

an

the

of

reagent

the

DEAE-cellulose

a kinetic

after

acid

as

preparation

and

hours,

reaction

showed:

B or

pooled

detected

The

analyz’er.

the

25OC

of

oxidation

Four

acid,

analysis

fractions.

this

was

of

showed

were

after more

digestion.

sample

the

by

carboxypeptidase

serine

performic

monitored

spectra

1 mg of

ami ?o-acids.

a column

end-terminal

material,

Approximately treated

the

N-terminal

were

contamination

using

by

performic

analysis

ultraviolet

Quantitative

accomplished

ml

by

and

inc-eased

of

300

identified

and

soluble

analyzer,

chains.

was

content

little

with

evaporation

collected

Water

(12:3:5).

polypeptide

portion

amino-acid

the

three

A substantial

Fractions

butanol-ammonia

buffer.

amino-acid

was

reacted

to

chromatography,

technique.

the

applied

paper

pair

acid-water

M borate.

was

solvent

automatic

COMMUNICATIONS

by

Y phosphate

thew-chymotrypsin

were After

1.5

the

0.1

material water.

Stein

connecting

O°C.

by

,a Phoenix

and of

and

RESEARCH

separated

butanol-acetic

on

Moore

o(“-chymotrypsin

taken

with

done

also

separated

NH3)

using

was

bridges

hours

was

separated

degradation

disulfide tovine

0.1%

BIOPHYSICAL

were

being

with

of

AND

DNP-amino-acids

derivatives

(n-butanol

the

BIOCHEMICAL

the

a mole = 0.5.

was

blocking

and

the

Phoenix value

of The

1, diffi-

Vol.

21,

culty the

No.

BIOCHEMICAL

5, 1965

encountered A-chain

in

hydrolyzing

indicated

that

-Pro-Val-Leu-Ser-Gly-leu. terminal

sequence

terminal

amino-acid

the

sequences

and

Meedom

combined

found

AND

the

Keil,

bond

connecting

This

sequence

agrees

with

with

the

data

on

total

the

sequence

(1963),

the

shown

giving that

the

remainder

a sequence

Hartley.

of

amino-acid in

Hartley

to

(1964),

Sot-m,

Acta,

jI8:559

et

also al

1.

:

h:

Cys-Gly-Val-Pro-Ala-Ile-Glu-Val-Pro-Leu-Ser-Gly-Leu

Hartlev:

Cys-Gly-Val-Pro-Ala-lle-Gln-Pro-Val-Leu-Ser-Gly-Leu :

Cys(gly,val,pro,ala,ile,gln)

Pro-Val-Leu-Ser-Gly-Leu

REFEREKES

Keil, B., Prusik, Z., Hartley, B.S., Nature, Meedom, B., Biochim. Sorm, F., Holeysovsky, Chem. Coma. a:2103

and Sorm,

F., Biochim. Biophys. m:l248 (1964). Biophys. Acta, a:429 (1958). V., Mikes, 0. and Tomasek, (1965).

496

V.,

Collection

C-

and

Cys-Gly-Val-Pro-Ala-Ile-Val-Pro-G1n-Leu-Ser-Gly-Leu

*ser-Waoner

of

content

1, which

Table

of

This

(1958).

TABLE Ueedom

proline,

with

COMMUNICATIONS

valine

bond

et al

was

RESEARCH

this

establishes by

BIOPHYSICAL

(1963).

Czechslov.

N-

shows

(1965)