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).