Introduction of sulfhydryl groups into macromolecules

Vol. 1, No. 3

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Sept. 1959

INTRODUCTION OF SULFHYDRYLGROUPSINTO MACROMOLECULES Irving

M. Klotz end Virginia

H. Stryker

Department of Chemistry* Northwestern University Evanston, Illinois Received

A

August

24,

1959

novel method has been described

1959) for the introduction into proteins

(Klotz and Heiney,

of mercaptan and acetylmercaptan

groups

based on the reaction: Q13CO-S-CH-CONH-PROTEIN

cH3co’s’cH-c~o + H2N-PROTEIN-j

h2COOH

;H2-C,c" 0

(II) cl)**

+ HS-CH-CON&PROTEIN

(I) In contrast

recently

to other methods (Sch(Jberl,

(III) 1948; Benesch and Benesch,

1958) for

the de of thiol groups into proteins, -- novo insertion which are based on the use of thioester or thiolactone reagents, that of equation molecules

(1) using an anhydride

containing

Mercaptosuccinyl forward

application

primarily

can be extended to macro-

hydroxylic

functional

polymers are obtained of the procedure

example, to a 5% solution

of dextran

* This investigation ~3 assisted Science Foundation..

readily

groups. by straight-

used with proteins. adjusted

For

to pH 8, one adds

by a grant from the National

** Compound (III) appears during the course of the reaction due to hydrolytic cleavage of acetyl groups from compound (II). This hydrolysis probably occurs in the vicinity of the droplets of NaOH added to maintain the pH. In reaction (1) amide formation has been represented to occur with the ipper anhydride bond of compound (I). This choice is based on the relative inductive effects of substituents described in L. P. Hammett, Physical Organic Chemistry, McGraw-Hill Book co., Inc., New York, 1940, pp. 211-12. 119

Vol. 1, No. 3

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

cH3co-s-cH--c~o ,0 +HO-MACRC?4OLECULE+ I CH2-C*o (I)

solid

acetylmercaptosuccinic

a period

ic addition conclusion IRA

+ (2) HS-CH-COO-~~ACROKOLECULE I CH2-COOH (V)

hour.

(I)

is maintained

of the reaction, 400 (in its

stirred

by dialysis

er, Amberlite removing

MB-l.

form) to remove hydrolyzed

salt were followed;

was the same. To recover the desalted

solution

Some typical The dextrans

charide interest,

4% content

with dextren

that a polysacIt seemed of

macromolecules,

with the third major For this the nucleic acids. acid was used..

anhydride

The

proceeded readily.

polymer are also shown in Table I.

There is no apparent therefore,

end lyophilized.

to attempt the reaction

for this thiolated

ed to biological

in each product

from Abbott

been dialyzed

could be thiolated,

with acetylmercaptosuccinic

Some results

for

polymer as a solid,

samples obtained

purpose a crude commercial yeast nucleic coupling

salts are

are assembled in Table I,

with dextran demonstrated

as well as a protein

class of biological

anhydride

both procedures

the mercaptosucctiyl

that marked -DL having

therefore,

Amber-

is lyophilized..

results

The experiments

the

At

a mixed-bed exchang-

preparations,

the total

used were clinical

Laboratories,

Thereafter

or passage through

For several

An at-

is passed through

which is not coupled to the macromolecule. removed either

by period-

solution.

above the solution.

the solution

chloride

in small amounts over

The pH is maintained

of NaOH to the continuously

mosphere of nitrogen lite

CH3cO-S-CH-COO-~Ji'~CRO~OLECULE I CH2-COOH (IV)

anhydride

of about one-half

Sept. 1959

reason why reaction Experiments rmcromolecules. Excellent

with polyvinylalcohol. 120

(2) should be limitwere attempted, preparations

of mer-

Vol. 1, No. 3

BIOCHEMICAL

captosuccinylated

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

synthetic

polymers were obtained

ent samples of commercial polyvinyl sic viscosities, tively

(Table

alcohols

Sept. 1959

with two differ-

of different

Du Pont ELvanols grade 72-60 and 71-30,

lntrin~ respec-

I), Table I Mercaptosuccinylated Macromolecules Moles (I) Added Moles Introduced per 105 GramsPolymer* per 105 Grams M t AoetylmercaptoPolymer s~zy&) succ*l(Iv)

Macromolecule DextrsA

600

De&ran-DL

1%

22 8

25 16

18

300

1

Polyvihylalcohol

72-60

600

20

5

Polyvinylalcohol

72-60

30

9

12

Polyvlnylaloohol

71-30

300

7

18

Bibonuclelc acid

* The mtrles in the third column refer to titration on the desalted preparation. Figures in the fourth column wre computedafter a similar titration was carried out on a sampleexposed to 0.01 g NaOH to remove the C&CO- llnked to the mercapto groups. From this latter titration we subtracted the numberof free mercaptan groups originally present.

In all the content metric

silver

several

of these preparations, of thiol

groups wxs determined

titration

well.

est range.

calorimetric

Ayers, Ho, Horowitz

Except for preparations agreed

by amperoIn

method with a disulfide

and Heiney,

with high cantat

Discrepancies

1958) was also used. of -SH, the two methods

as high as 20% were found at the high:

Since we are uncertain

in the upper ranges,

primarily

(Benesch, Lardy and Benesch, 1955).

cases a quantitative

dye (Klotz,

as well as those of proteins,

which method is more reliable

only the amperometric

results

are listed

in

Table I.. For dextran was carried

and polyvinylalcohol

out to verify

to the macromolecule.

an ultracentrifugal

that the thiol An azomercurial 121

group is actually dye which reacts

test linked specific-

Vol. 1, No. 3

ally

BIOCHEMICAL

with mercaptan groups (Horowitz

to a 1% (buffered) rapidly this

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

solution

solubilized

solution

and Klotz,

insoluble

The schlieren

moved together.

polymer,

of

in a Spinco Node1 E

maximum end the color

With mercaptosuccinyl

which

dye.. A portion

was sedimented at 60,000 r.p.m.

ultracentrifuge.

1956) was added

of mercaptosuccinyl

the normally

Sept. 1959

boundary

polyvinylalcohol,for

ex-

ample, a sedimentation coefficient of 1.93 S was computed from the schlieren patterns and a value of 2 S 2 0.5 S was estimated from absorption color.

optics.

Furthermore,

Consequently

the thiol

macromolecules

in solution.

out with the nucleic separating

hydrolyzed

liquid

groups must be attached Similar

experiments

acid derivative,

showed no to these

were not carried

but since the methods for

compound (I)

with those used for dextran nucleic

the supernatant

from polymer were identical

and polyvinylalcohol,

acid must also have covalently

the modified

bound mercaptosuccinyl

groupsThus a very of thiol cule,

simple method is available

groups into

as well as into

groups can be readily cross-links

all

three classes of biological

certain oxidized

synthetic

on the configuration

polymers.

Since disulfide

role in protein

to examine the effect

and behavior

macromoleThese -SH

to -S-S- linkages.

play such en important

should be very interesting

for the introduction

molecules,

it

of such linkages

of these other

classes of macro-

molecule. BIBLIOGBAPHY B. E. Benesch, H. A. Lar.dy and R. Benesch, 6. Biol.

Chem., &,

663 (1955). R. Benesch and R.

E.

Benesch, Proc. Natl.

&&

&&,

44, 848

(1.958).

M. G. Horowitz

and I. W. Klotz,

&&.

(1956). 122

Biochem.

BiODhYS.,

a,

77

Vol. 1, No. 3

BIOCHEMICAL

I. M. IUotz,

J. Ayers, J. Y. C. Ho, M. G. Horowitz

R. E. Heiney, I. M.

IUotz

A. SchUberl,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and

Sept. 1959

and

2. Am. Chem. So&, 80, 2132 (1958).

R. E. Heiney, 2. && &gg.

Anaew. Qg&,

63, 7 (1948).

123

&&,

&

3802 (l-959).