Covalent chromatography — The isolation of tryptophanyl containing peptides by novel polymeric reagents

BIOCHEMICAL

Vol. 70, No. 4, 1976

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

COVALENT CHROMATOGRAPHY - THE ISOLATION

OF TRYPTOPHANYL

CONTAINING PEPTIDES BY NOVEL POLYMERIC REAGENTS

by Menachem Department

Rubinstein,

of Organic

Yoram Shechter

Chemistry,

The Weizmann

Rehovot, Received

April

and Abraham

Patchornik

Institute

of Science

Israel

29,1976 Summary

Specific polymeric reagents for reversible covalent binding of tryptophan residues have been developed. Polymers bearing Aryl-SxCl groups (x=2-3) were prepared by binding thioaryl groups to cross-linked polyacrylamide, and subsequently reacting the products with an excess of S2C12. The resulting polymers were allowed to react with various mixtures of amino acids and peptides (excluding cysteine and its peptides) in acidic media. It was found that tryptophan as well as tryptophan-containing peptides were selectively bound to the polymer. Upon reduction with thiols (e.g. dithiothreitol), 2-thiotryptophan or its peptide derivatives were cleaved from the polymeric matrix. The proposed method is used for a one step isolation of tryptophanylcontaining peptides from peptide mixtures as well as for introducing thiol groups at the tryptophanyl residues. Introduction The study partial

of protein

degradation

peptides.

and time-consuming

thioproteins, This

type

polymeric covalent mild

Copyright All rights

it

has been

of chromatography

binding

protein

desirable

in many cases

molecule

and separation

processes to develop

are laborious simple

and

methods.

which

reagent

requires

separation

is

of such an improved

and selective

polymeric

or modified

Conventional

and therefore

separation

An example

and function

of the native

of the resulting

effective

structure

introduced is

based

to one of the to the polymer. method

method

for

covalent

by Brocklehurst on the specific

components In addition,

releasing

it

1257

chromatography

is

of

and coworkers affinity

of a mixture

the bound

matrix.

0 1976 by Academic Press, Inc. of reproduction in any form reserved.

is

of an insoluble

resulting essential

component

(1).

in its to have

from the

a

BIOCHEMICAL

Vol. 70, No. 4, 1976

Arylsulfenyl reagents media It

for

chlorides the

to form

be a useful

and sulfur

selective

tool

The preparation

the rapid

of these

residues

and their

peptides

are

use for

isolation

covalent

demonstrated

residues

analog

their

specific

at acidic

reagents

(2,3)

might

containing

reaction

chromatography

useful

respectively

of these

of tryptophan

in the present Materials

are

and 2-thiotryptophan

polymeric

polymers,

(S2C12)

of tryptophan

tryptophan

an insoluble

for

monochloride

modification

2-arylsulfenyl

seemed to us that

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

peptides.

with

tryptophan

of tryptophanyl-containing

study.

and Methods

Glucagon was obtained from Sigma Chemical Co., human serum albumin (HSA) was purchased from Mann Research Laboratories. The standard calibration mixture of amino acids type I (which does not contain cysteine) was purchased from Spinco-Beckman and an equimolar amount of tryptophan was added to it. This solution was evaporated to dryness at room temperature under vacuum and redissolved in the appropriate buffer. Polyacrylamide, Bio-Gel P-10 and P-200 were the products of Bio-Rad Laboratories. The peptide Z-Tip-Ala was purchased from Miles-Yeda (Rehovot, Israel). Other chemicals were obtained from Fluka A.G. Amino acid analyses were performed essentially according to Spackman, Stein and Moore (4), Tryptophan was determined by using a 15 cm column at pH 5.25, it had an elution time of 45 min. (lysine was eluted after 60 min.). Z-thiotryptophan was identified by TLC on silica gel plates using a n-butanol-acetic acid-water (45: 05:125v/j) solvent system (3% dithiothreitol was added). Preparation of polymer B. Polyacrylamide-acyl azide (5) was reacted with a solution of 4,4’diaminophenyl disulfide and triethylamine in formamide. The resulting polymer (Fig. 1) was isolated by filtration and reduced with aqueous NaBH4. The dried product (A) was suspended in 10% solution of SzClzin dimethylformamide giving polymer B which was isolated by filtration and washing with CSz and dimethylformamide immediately before use. Preparation of polymer D. Aminoethylated polyacrylamide (5) was reacted with a solution of the di-N-hydroxysuccinimide ester of S,S’dithiobis-2nitrobenzoic acid (6) and triethylamine in formamide. The resulting polymer (Fig. 1) was reduced with NaBH4 and treated with SzC12 as described in the preparation of polymer B. Results Reaction typical

experiment

1.0 ml solution of tryptophan suspension off.

of the polymers polymer of amino was added

was stirred

Analysis

of the

with

amino

acids

B (20 mg) or polymer acid

calibration

(1 nmoles for filtrate

calibration

D (100 mg) were

mixture

of each

amino

to which acid

30 min at room temperature have shown

the presence

1258

mixture.

In a

suspended

an equimolar

in 2% acetic

of all

amount

acid).

and the polymer amino

in

The was filtered

acids

(loo+

3%

BIOCHEMICAL

Vol. 70, No. 4, 1976

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

CON3 t (H2NaS-) A

-

j-CONHC2114NHtO

02NxLzCI

0 (X*2-3

of the

starting

filtrate.

amount)

Similar

acetic

acid.

water

Next,

dithiothreitol

except

results

in

of z-trp-ala

Polymer

(1 Rmol)

in

then

filtered

off.

The decrease

Next

and finally

suspended

(0.7

suspended

10% aqueous

20 nl aliquots of the

was washed

of polymer

filtrate

with

with

for

buffer

shown the

acid

(1 umol)

of each amino

The polymer

solution

(from 7 O.D. removal

of acetic

acid

was

acid

and water

The filtrate

found

(10.5

umol)

at intervals,

at 280 nm was measured.

2.

1259

Polymer in

filtered

was alanine

B (50 mg) was

1 ml of 2% Aq. acetic off

The results

and the are

to

of

of the parent peptide). tryptophan.

and

30 min in a solution

(see above). amino

acid

was detected.

a quantitative

an excess

of

0.05M

of amino

(1 ml).

of the

The only

B with

of L-tryptophan removed

acid

in absorbancy

1 ml of stripping

were

(Rf=0.46)

the presence

acetic

agreement

of 10% acetic containing

mixture

from the

Q-100%)

TLC of theeluatehave

calibration

to 70% recovery

rate

(pH 8.5)

no tryptophan in

removed

concentrations

an excess

30 min.

and analysed.

in a solution

absorbance in Fig.

in

corresponding

The reaction

acid.

the polymer

acid-hydrolysed

nmol,

acid

at 280 nm) was in

Z-Trp-Ala.

with buffer

Z-Trp-Ala

and amino

tryptophan)

was then

for

B and D.

was completely

D (100 mg) was suspended

except

units

was washed

D with

reagents

at various

(Rf=0.24),

acid

1.4 O.D.

obtained

buffer)

of polymer mixture.

which

1 ml of O.lM Tris

of 2-thiotryptophan

calibration

tryptophan

were

(stripping

Reaction

of the polymeric

the polymer

and suspended

presence

I

The preparation

Fig.

8(x=2-3)

given

BIOCHEMICAL

Vol. 70, No. 4, 1976

I 2

Fig 2.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I 4 Time

I 6

I 6 (mid

I ,t IO”90

I_

The rate of reaction of polymer B with tryptophan.

The react'ion of polymer B with a peptide mixture derived from human Humanserum albumin (30 mg) was digested with trypsin

serum albumin.

described elsewhere (7).

The mixture was lyophilized

as

and dissolved in formic

acid (0.3 ml 98-100%). Polymer B (60 mg) was added and the mixture stirred 1 hr.

The polymer was then filtered

water, 8M urea in O.lM Tris buffer covalently buffer

bound peptides.

off,

(pH 8.5) and water.

of the extract

positive

Val

A

sample of the extract

was applied

(pH 3.5) which have shown the presence of

spot (moving towards the anode).

Another sample

was acid hydrolysed and analysed for its amino acid content.

The amine acids found were: Arg (l),

This removes the non-

The polymer was then suspended in a stripping

to high voltage paper electrophoresis

tryptic

washedwith an excess of formic acid,

(2 ml) for 30 min and then removed.

only one ninhidrin

for

Arg (1.05),

val (0.9) and Ala (2) [Theoretical:

Trp (1)

(1) and Ala (311 corresponding to the single tryptophan containing

peptide of humanserum albumin.

This peptide was recovered in 40%

yield. The reaction

of polymer B with a peptide mixture derived from glucagon.

Glucagon (6 mg) was digested with trypsin lyophilized,

for 6 hr.

The reaction mixture was

dissolved in formic acid (0.3 ml) and treated with polymer B

(50 mg) for 1 hr.

The polymer was then filtered

1280

off and washedwith excess

BIOCHEMICAL

Vol. 70, No. 4, 1976

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

-HNYco-

.w---HNCHCOCC,

-HNFHCo-

oq. ACOH +CON”++=A;~@

B+

b3 H

(x.2-3)

Fig 3.

The reaction of the polymeric reagents with tryptophanyl derivatives and the release of 2-thiotryptophanyl derivatives by thiols.

of formic acid, water, 8M urea and water. 1 hr into a stripping

buffer

lyzed and analysed for its Arg

(I),

Met (l),

amino acid content.

[Theoretical:

Leu (1) Phe (l),

containing tryptic

A sample of the extract

Thr (0.951, Glu (2.15),

Asp (2.1),

Leu (0.92) and Phe (0.94). Val (l),

(2 ml).

Next the polymer was suspended for was acid hydro-

The amino acids found were:

Ala (L), Val (0.95), Arg (l),

Trp (l)].

Met (0.9),

Asp (2), Thr (1) Clu (2), Ala (l),

This peptide is the singletryptophan

peptide of glucagn. Discussion

The use of polymers as chemical reagents has been recently Covalent chromatography is a relatively

reviewed (8).

new use of polymeric reagents and the

present work is an addition to the few examples known in literature. of S2C12 with thiols

is described in the following S2C12+ RSH4

The intermediate

polymeric backbone in a rather high concentration this is another example of the ability

equation (9): RSSSSR

[RSSSCl] -

RSSSClhas not yet been isolated.

of isolating

The reaction

We have isolated

it on a

(0.8 mm01Cl/g of polymer B) and unstable intermediates on a

polymeric support. The reactivity

of polymers B and D towards tryptophan

and tryptophan-

containing peptides (Fig. 3) is analogous to that of S2C12 and of o-nitrophenyl-

1261

Vol. 70, No. 4, 1976

sulfenyl

BIOCHEMICAL

chloride

tryptophan

which

respectively.

be bound

When peptide

could

adsorption

be removed

removal

of the

reaction

with

peptide

by exhaustive bound

thiols.

mixtures.

was inserted This

covalent

(7),

but

and the higher

it

which

could

that

by this

Fig.

of the polymeric

However

8M urea.

The

achieved

can be isolated a thiol

by from

group

3).

to affinity because

was

in turn,

was then

process

(see

comparable

These,

and with

peptides

tryptophan

it

to the polymers.

acid

is more favorable

capacity

clearly

reagents

observed.

tryptophan

of the is

bound

formic

be mentioned

chromatography

peptides

preparation

chromatography of the

reagent

of

simplicity

compared

of

with

the

column. Selective

isolation

is of considerable view

should

tryptophan

from the polymers

process

at the 2-position

tryptophan

affinity

peptides

By this It

with

have

the polymeric

was also

washing

tryptophan

were

peptide

mixtures

of polymer. with

peptides

of other

acid towards

an excess

had been reacted

tryptophan-containing

non-specific

amino

reagents

by applying mixtures

or 2-o-nitrophenylsulfenyl

with

of the polymeric

quantitatively

that

2-thiotryptophan

Our results

shown the specificity

found

give

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of tryptophan-containing

potential

of the relatively

modifying

residues

This

work

National

of Health.

from protein

sequences,

particularly

of tryptophan

reagent

in native

AcknowledgementInstitute

in protein

low abundance

use of the polymeric of tryptophan

utility

peptide

for

proteins

probing is presently

was supported

by Grant

residues the

in

in proteins.

differential under

digests

The

accessibility

investigation.

No. AM05098

from the

References 1.

Brucklehurst, K., Methods Enzymol.,

2.

Scoffone,

3.

Wieland,

Carlsson, J., 34, 531-544.

E . , Fontana, T.,

Weinberg,

Kierstan,

M.P.J.

and Grook,

E.M. 1,

A. and Rocchi,

R. (1968)

Biochemistry,

0. and Dilger,

W. (1955)

Ann. 592,

1262

69-80.

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Vol. 70, No. 4,1976

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

4.

Spackman, D.H., Stein, W.H. and Moore, S. (1958) Anal. Chem., 30, 1190-1206

5.

Inman, J.K. end Dintzis,

6.

Shechter, Y., Rubinstein, M., Becker, R. and Bohak, Z. (1975) Eur. J. Biochem., 58, 123-131.

7.

Wilchek M. and Miron, T. (1972) Biochim. Biophys. Acta, 278, l-7.

8.

Patchornik,

9.

Feher, F and Kiewert, E. (1970) Z. Anorg. Allg.

H.M. (1969) Biochemistry,

8, 4074-4082.

A. and Kraus, M.A. (1975) Pure App. Chem. 43, 503-526.

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