The basic trypsin inhibitor of bovine pancreas V. The disulfide linkages

Vol.

20,

No.

AND BlOf’HYSlCAL

BIOCHEMICAL

4, 1965

BASIC TRYPSININHIBITOROFBOVINEPANCREAS V. THE DISLILFIDELINKAGES* Beatrice Kaeeell and H. Laekoreki, Sr.+

THE

Leboratory for Cancer School of Medicine, Milwaukee,

Biochemicel

ReceivedJuly2, Recently penczuatic

ua

Chuvet

present

experiments

ture of

the

presented

the

inhibitor et

al.

University

Marquette

Uieconein 53233

which loaete protein. 3

4

5

sequenceof

linear

(Keeee~l nd

acida

meino

in

the

Laekowki, 1964; Kaeeell

basic et

al.,

a slightly different l equenca. original maulte (Fig. 1) end eumazizee

(1964)

confirm

p-8’:

12

Reeearch,

1965

trypein

1965). The

RESEARCH COMMUNlCAllONS

published our

tha

5-5

6

linkages

7

8

to

9

coaplate

lo

the

priory

etruc-

11 12 13 14 15 16

ARG-PRO-ASP-PHE-CYS-LEU-GKl-PRO-PRO-TYR-Tt#?-GLY-PRO-CYS-LYS-ALA17 16 19 20 21 22 23 24 25 ARt-ILEU-ILEU-ARG-TYR4IEE-TYR-~-A~-LYS~~~LY-~U~Y~~-T~-

26 27 28 29 30 31 32

33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 PHE-VAL-TYR-GLY-GLY-CYS-ARG-ALA-LYS-AR6-A!jN-ASN-PHE-LY!%!XR-ALA-

49 50 51 52 53 54 55 56 57 58 GLU-ASP-CYS-MET-ARS-THR-CYS-GLY-GLY-ALA

Figure

Linear

1.

Becauseof conditions (Ryle

the

of

Sequence

the

Pancreatic

Trypein

l enaitivity

and

of S-S linkegea to axchmge Senger, 1955; Ryle rt. 1955; Speck-

Inhibitor

under et

alkalino ml. 1960)

end the undesirability of using *my enzyme containing -SH groupe, we were unable to digeet the native inhibitor. It is raeietant to ell the comon (Kaeeell end Laekweki, 1956, 1965). Two methods were used obtain S-S peptideet enzymic digaetion of the euccinyleted inhibitor l cid hydrolysis. Methods of l nalyeie were derribed previously.

enzyme

: ated

by

the

procadwam

This work from tha Nationel Science l

AM 00535 +

Amarican

of

Hebeab

The

Society

and

inhibitor (BB micraolee) wae euccinyl(1956) at pH 8.0. The pzoduct use

=Q&.

wee supported by Public Health National Inetitutae of Health Foundetion. Center

to

Research

463

Professor.

Sazvice end Crant

Reeaerch G-7501

tint

from

the

Vol. 20, No. 4,1965

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNlCATlONS

TABLE

I

Ratioa of Amino Acida in 2 Paira of Oxidized Peptidaa Tram the Enzymic Diguata ot the Succinyl Inhibitor (Impuritiaa era shown in parentheses)

Molar

-

?

.r 2

A

B

Alanina

(0.11)

1.00

Aapartic Acid Cyataic Acid

mot) 1.00 1.04 (0.11)

1.02 1.06 1.12

tlutamic

Acid

Glycine Laucine Lyaina

(O.OT) (0.02)

(0.05) (0.01) 0.90

hthionina Sultana Phanylalanine Serina

(0.04)

0.93

Ol.32)

(0.02)

(0.04)

Thraonina Valina

0.89

0.84 (0.12)

B 0.94

(0.04)

(0.21) 1.20

1.00 1.07 (0.19) (0.09)

1.24 1.00 0.91 0.73

(0.03) 0.92

0.57

(0.07)

(0.19) 1.05

(0.06)

(0.15) --

(0.21)

I 9 8 71 6a 5 4

100 200 300 400 530 600 TUBE

NUMBER

TUBE

NUMBER

Fig. 3 digest of the auccinyl Figure 2. Chromatographyof the peptic-tayptic inhibitor on phoaphocelluloaa. Column2.5 x 24 CII. fractiona 8.5 ml. Rate 60 ml./hr. Starting b&tar 0.02 H Pnanium ecetete, pH 3.95. (i-radiant started at once* Elution curve. - - - - pH. Arreua above peaks indicate positive teat for S-S. Figure 3. Chromatographyof tha partial acid hydrolyaate on phomphocelluloaa. Colum 1.9 x 45 cm. Fractions 4 ml. Rata 35 rl/hr. Starting buffer, 0.02 H amaenium acetate, pH 3.85. Gradient started et arrow on baaaline,. Elution curve. - - - - ptl. Arrows above pa&a indicate positive teat for S-S. Fig.

2

464

BIOCH~ICAL

Vol. 20, No. 4, 1965

It

inactive. at pH 1.5 et pH 3.5

vae

et

a concentration

of

10 mg/ml

with

and

chronatographed on phosphocalluloas (Fig. 2). were those used by Canfiald end Anfinsen (1963).

pH 3.4

and

buffers

individuel

The

diagonal

paper

(1963).

Two

both

derived

They

8ccounted

0.0s

pepain

and then digested with trypain SP for 40 hours, lyophilized, 370 for 96 hour8 (inhibitor concentration 17 mg/ml, tryp8in to 115 ml, adjusted to 0.15 mg/mlI. The digest was diluted

and

concentration

Their

dig8eted

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

which conteinad cyetina and oxidation method

peak8

8lBCtrOphOr8t3is

pairs

of peptides, the last part

from

togethar

on

derived

&ructurss

a

from

both

of

containing

th8

for 1.7$ in Teble

smaly~es

procedure

subjected

of

Brawn

same

break-through

the

molar basis the

w8re

The

to md

S-!?

and

the

Hartley

linkage,

and

peak, were identified. of

the

protein

digastcd.

I wre:

30 C:5-GLN-THR

IA I.!3

LYESER-ALA-GUI-ASP-CYS-MET 51

30 LYS-AU-GLY-LEU-CiS-W-THR4nE

2A

ASP-CY!sET

28

51 The

other

peaks

contained

very

which was obtained feirly ing the othar 2 %g linkagea,

0n8

51,

confirmsd

linkage

the

large psptidas pure

yielded

and

which,

satablishsd

with

more

than

one

S-S

on oxidation three piecea containby the absence of cyatinee 30 Md

above.

5 14 CYS03H-LElJ-GLU-l+0-F’RO-TYR-TtWLY-PRO-CYSO3H-LYS-ALA-ARG-ILUJ 38 VAL-TYR-GLY-GLY-CYSO3H-ARC-ALA-LYS-AREASN-ASN 55 AR&THR-CYS03H-GLY-GLY-ALA

ski, with

partial

Partial acid hvdrolvqt 1964) on digestion of 0.03 H MC1 under the

Our previous the

N-terminal

conditions

linkage.

3A 3B 3c

crxperimmta

(K8~~aell and

pentadecapaptide of

Schultz

et

al.

of (1962)

th8

Laakov-

inhibitor

showed

that

cy8tint 5 and cy8tina 14. 'ye therefOr0 usad the some&at more drutic conditions of McDowell and Smith (1965): q icnmmlas of inhibitor, cont. 5 mg/ti, 0.03 M MC1aaturatad with N2 end containing 1.1 x lOA t4 thioglycolic acid, logo, 46 hours, Dealed evacuated tubea

8plitting

The

(Fig. 3). reason&ly

occurred

digsmt From pur8,

uaa

between

lyophilired

and

chromatographed

on pho8phocslluloes

fine1 peak, which umm elutad betwaen pH vary basic peptide wee ieolated in a yield,

the

465

6 and ~a a

a molar

6.5,

50

BIOCHEMICAL

Vol. 20, No. 4,1965

AND B0PHYSlCAL

RESEARCH COMMUNICATIONS

TABLE II

Molar

Ratio Hydrolysat~

of

Asia0

Acide

of en 5-S Paptide from the Pieces Sspustsd sftsr Oxidation are shown in psrsnthseer)

Acid

and ot It8 (Impurities S-S

Oxidized

Psptids

piace

Oxidixed

4A

piece

Alanins

2.57

1.15

Arginina

3.90

2.02

2.33

(0.291

(0.08)

(0.11)

Aspartic

Acid

4B

0.88

1.78

Cyrtin8

Cyatmic

1.00

Acid

6lutamic

Acid

Glycin8

(0.37) 2.00

(0.05 1 0.85

l.Of

1.01

Iaolwcinm Leucins

(0.39)

Lysine

2.17 (0.17)

Mathionina Me%hionine

1.00 0.88

(0.03)

1.06

1.21

SW

Sulfom

0.80

1.19 1.21

Phenylalsnine

Proline Thrsonine

1.03

(0.87)+, 1.39

Tyrosim V8lin8

1.01"8 (0.M)

(0.41)

* Attar 24

(0.08)

(0.38)

--

hydrolyaia, the usual value for tha 2 ioolwcina (#la end 19) ia 1.0-1.3. The threonine appeared as 8 whole ro@lue here, but not in the Yhilo,it sight be mhl~~s #ll, it ia mre likely to pieces. For 2 r88idu88 in the psptids prsaant M irpurity. Plus somaoxidized tyrosine.

rsoidues

of

* repsrated recount

-* basis,

of

l tructum

this

hours protein

Analymia (Table II)

12$ of tha starting 88tsrial.

indicatad the

1 4A

6LY-PRO-CYS-LYS-ALA-AR&ILW-ILEU-AR6-NR-PHE-NR

4B

GLY:MiZUYS-AR.

38 The

peptide

ths

pieces

warn oxidized

tha

expoctad aaino

sspsrstsd

with by

r&d

porforaic

acid

according

ela8tropborsaia.

pqer colpositim

(Tsbls

The

II).

to

Fo-ly,

alkalinm conditima during elutio~ could nctth8vaewaad bwsuso

this The

was

ths

only

linkage8 split

psptide by

0.03

remaining

on the

Hirs

individual

(1956)

md

pin88

h8d

oxpow

S-5

intorehangs

COW.

t4 HCl to fom this psptida won of tba

466

to

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 20, No. 4,1965

type

(1950) and

and

10~.

ARG-ASN The

of

fra

expected

the

structure,

cit.):

TM-SLY

of

thin

(U-12),

(Partridge and Davis

m&hod

TYR-ASN

(23-241,

GLY-GLY

S-S

linkage ie therefore 5-55 and the

cmrplate

location of the S-S linkages indicates which

is

in

l gramnt

a

compact, interlocked

the properties

with

of

21, reversed GIA-31 end TM-32 and tentatively miaplacad tailsd degradation of the tryptic paptida, 40-42, wee

this

protein,

ARG-42 3 amil

already

(Kaeeell and Laekmaki, 1964). Our aequancause confirmed paptidea 33 and 48 above. Table XII shove the abtrrtiva

blar cM-cys

by

Degradation

Ratio

of

of

Peptide

30-33

Amino Acida Remeining !;

GLU

THR

PHE

NH3 1.02

Free

1.00

0.0s

0.97

0.92

1.ao

-

-

0.13 s-

0.92

1.00 1.00

-

6d -

acid

by

0.16 ftea

the iaoletion dagsada-

Aeino Acid from PTH

0.92

+ 4e recovery as . . . .

De-

(US-GLN-THR-PHE)

a.93

3

groups.

Edmn

o.t4

2

capouncl

Edmen

poaition

III

TABLt

Subtractive

in

praaantad

of

0 1

structure

is;

especially its great raaiatanca to enzymic digestion. saou%nc6confixnationr Chauvet at. (1964) placed

SW

(3637)

(42-43).

rsndning

inhibitor

The

l tudiaa

pruvim~a

glutmic

extzactad.

467

B-s

acid

bdrolyaia

of

the

PTH

Vol. 20, No. 4, 1965

BIOCHEMICAL AND BlOPHYSlCAL RESEARCH COMMJNfCATK)NS

tion of tha chymotryptic paptide, X3-33, the tryptic peptide 18-20, the molar ratio 0.89

to

1.W

a&ter

24 houra

hydrolyaia

and

to

confirm of 1.55

our

ieolaucine to

1.00

31-32 to after

sequence.

was

arginine 72

In

houra

hydrclyaia. Thia indicetaa only 1 arginine in this position pracsded 2 isolaucinaa. Analyaia of the overlapping peptides previously ahoun

by

(Kaasell et al. 1965) for aamvmiagave at lasst 2 and aa manyas 7 separate end unaquivocel determinations of the presence or absence of aide groups for awry aapartic or glutamic acid in the hydrolysates.

REFERENCES Brown, Canfisld, Chauvst,

J.

R. and Hartley, B. S., A. E. and Anfinaan, C. J., Nouvel. t. end &her, (1964). H. Habsab, A. F. S. A., Casaidy,

Biochem. B., J. R.,

J., E (1) 59P (1963). Biol. Chew, 238, 2684 (1963). Biochim. Biophya. Acta 9& 200

6. and Singar, S. J., Biochim. Biophya. Actajj,& 587 (1958). Hbs, C. H. W., J. Biol. them., 219, 611 (1956). Kaaaall, 8. and Laakowaki, PI., Sr., Eiocham. Biophya. Reaaarch Coasnun., & 255 (1965). Kaaaell, B. and Laskouaki, R., J. Mol. Cham., a, 203 (1956). Kaaasll, E. and Laakcuaki, H., Sr., Biochem. Biophya. Raaaarch Conwn., 11,

792

(1964).

Kaaaall, B. and Laskowaki, McDcnaJl, M. A. nd Smith,

H., Sr., Fsd. Pro&&, 593 (1965). E. L., J. Biol. Chem., 240, 281 (1965). S. H. and Dsvia, H. F., Nature &, 62 (1950). P., end Sanger, F., Biccham. J., &, 535 (1955). P., Sangar, F., Smith, L. F. end Kitei, R., Biochaan. J., &,

Partridge,

Ryle, A. Ryle, A. (1955). Schultz, J., Allison, Speckman,

D.

H.,

Stsin,

H.

and H.

U.

Grice, H., Biochemistry and Meore, S., J. Biol.

(1960).

468

A, 694 &am.,

(1962).

235, 648

541