Rapid Reduction
Reagents for
Bonds in Proteins
of Disul fide
Rajeeva Singh
ImmunoGen, Inc.
Cambridge, I4A 02139
Georgre M. Whites ides
Department of Chemistry, Harvard University,
Cambridge, MA 02138
I .
Introduct
ion
used in
are routinely
reagents
Disulfide-reducing
(i )
the
reducing
f or
manipul,ations
biochemical(ii1
proteins
and
in
bonds
native
disulfide
groups in proteins
thiol
the essential
maintaining
the
disulfide
to
oxidation
their
by preventing
(DTT) is the most popular
Dithiothreitol
state.
(1) .
however/
DTT is,
reagent
disulfide-reducing
The value of
at pH 7-8.
slow in reducing disulfides
(9.2) and
groups in DTT is high
pKa of the thiol
( - 1 %) o f
at pH 1 only a small- f raction
therefore
p
r
e
s
e
n
t
r
eactive
i
n
t
h
e
g
r
o
u
p
s
in DTT are
thiol
form.
thioLate
reagents
We have developed severa.l- new dithiol
(2-6) .
groups
disulfide
of
reduction
rapid
for
bonds by the
reagents reduce disulfide
These dithiol
(Eq 1) .
interchange
mechanism of thiof-disul-fide
r ssR
rsH
R'+
\sH
RSSR _
R'
+
\sH
RSH
-nlr
a?
\i
2RSH
(1)
reagents is based on two
The design of these dithiol
( i ) a l - o w v a l u e o f P K a ( - 1 t o B) o f
requirement s :
(i i )
reduct ion
groups
a high
and
thiol
t.heir
is influenced
potential.
of a thiol
The reactivity
T E C H N I Q U E SI N P R O T E I NC H E M I S T R YV I
c r e s sI.n c .
C o p y r i g hO
t 1 9 9 -b5y A c a d e m i P
in any fbrm reserved.
All rightsof reproduction
259
260
R a j e e v aS i n g h a n d G e o r g eM . W h i t e s i d e s
present
fractj-on
in the thiolate
form
both by its
A
and by the nucleophilicity
of the thiolate
anion.
group of 1ow pKa has a significant
fraction
thiol
present
in
react ive
f orm, but
the
thiol-ate
the
nucleophilicity
of its thiolate
anion is lower than
it is for a thiol
of higher pKa.
The overall
effect
is
rate
that
the
apparent
of
thiol-disu]
fide
interchange
is maximum for a thiol
whose pKa value
is approximately
equal to the pH of the solution
(3) .
groups have pKa
A dithiol
reagent whose thiol
val-ues of -1 to B and which has a high reduct ion
potential
is therefore
expected to reduce disulfide
bonds rapidly
at pH 7-8.
We have developed several new reagents
INrN'( D M H ),
dimethyl-N, N'-bis (mercaptoacetyl) hydrazine
(BMS) and meso-2,5bis (2-mercaptoethyl)sul-fone
(DTA) l
dimercapto-N, N, N' , N'-tetramethyladipamide
groups have pKa val-ues of -1 .B (2-6).
whose thiol
Based on Bronsted correlations
these reagents
are
groups at pF{ 1 faster
expected to reduce disul-fide
(3,5).
than DTT by a factor
of -5
This
report
focuses on the comparison of reactivities
of BMS/
DMH, and DTT toward
groups
disulfide
in
several
proteins
under nondenaturing conditions
at pH 1 .
Q"-sn
oT"qrsH
BMS
f I .
Materials
">-.
CONMe2
MeN
SH
SH
Vfef,f
SH
SH
\*/
.//
o'
*o)^r*
Ho."\,,sH
CONMe2
DMH
and
DTA
DTT
Methods
BMS and DTA are
from
avail-abl-e
US Biochemical
Corporation.
The synthesis of BMS, DMH and DTA are
straightforward
from readily
material-s (4available
Papain-S-SCH3 was prepared as described before
6) .
(1) .
(bovine
pancreas)
Trypsinogen
and Cx,-chymoA (bovine pancreas) were purchased from
trypsinogen
261
R a p i d R e d u c t i o no f D i s u l f i d eB o n d s
(IgGr)
Sigma. The murine monoclonal antibody anti-84
supernatants.
from hybridoma culture
was purified
BMS, DMH and DTA are sol-ids at room temperature.
(-10 mM) in
stock sofutions
We recommend that their
(50 mM sodium phosphate, pH J, 1 mM
phosphate buffer
to
sonication
in EDTA) be prepared fresh by brief
These solut ions can
ensure complete sol-ubilizat ion .
groups by Ellman's assay (B).
be assayed for thiol
A.
of
Reduction
BtrIS, DMH and
Papain-S-SCH3
DTT
Using
Samples of papain-S-SCH3 (0.042 mq/mL, r.B pM) in
(pH I | 2
deoxygenated 50 mM sodium phosphate buffer
(25
reagent
mM in EDTA) were reduced using dithiol
t
i
m
e
A
t
s
e
v
e
r
a
l
2
3
"
C
.
D
T
T
)
a
t
D
M
H
o
r
plM; BMS,
(7-, 6-, II- , L6-, and 21-min) , aliquot s
interval-s
mixture
were added to
reaction
(200 pL)
the
of
(
8
0
0
p
L
N
b
e
n
z
o
y
l
-L-arginine-pm
M
o
f
3
.
4
substrate
pH 6.3,
b
u
f
fer,
i
n
m
M
b
i
s
t
r
i
s
nitroanilide
50
1 mM EDTA and 5% v/v DMSO) and the rates
containing
in absorbance at 410 nm were measured.
of increase
was in excess over that
The concentration
of dithiol
assumed to be
of papain-S-SCH3, and was therefore
reduction;
the
of
during
the
course
constant
pseudounimolecular.
For the
is therefore
kinetics
(kupp)
reduction
by DTT, the apparent rate constant
-ln [ { (maximum
plot
of
was calculated
from
the
- (regenerated
papain
papain
activity)
regenerated
activity)
I vs
I / (maximumregenerated papain activity)
+u'ri r rmr as r
f! vn! r
reductions
^ ^ ! . i - - . 1 ! .
auL-LV-Lr-y
rw. rr hr !i ! .nrhl
using
wq.S
,qr rI \ ar rr rl sa
:
kupp
IDithioI
BMS and DMH, the
m e a^ ^ S, , -u^ .rl e d
ar { t-
' 1
l
- . i n
min
For
] .
regenerated
- i n
in
four
€ ^ " -
the
papain
S
e ph - cr r rf nr d L e
and kapp was cal-cul-ated using the rate
experiments,
-ln [ { (maximum
papain
regenerated
equation:
- (regenerated
papain
activity)
activity)
I / (maximum
regenerated papain activity)
I : kappIDithiol]t.
B
Reduction
I
Dithio
of
Trypsinogen
Samples of tryPsinogen
sodium phosphate buffer
Using
(5 mglml,, 0.2L mM) in 50 mM
(pH J | 1 mM in EDTA) on ice
262
R a j e e v aS i n g h a n d G e o r g eM . W h i t e s i d e s
(OoC) were reduced using dithiol
(0.5 mM; BMS/ DMH/
DTT).
At 10-, 20-, 3O-, and 200-min time intervals,
(200 l.ll,) of the
aliquots
reaction
mixture
were
purified
by gel-filtration,
and were anal-yzed for
thiol- content using El-lman's assay and for protein
concentration
by measuring absorbance at 280 nm (2) .
Under these conditions
a maximum of 0.6 disulfide
per
residue
was reduced
trypsinogen
molecul-e.
Assuming pseudounimolecul-ar kinet ics,
the apparent
(kapp) was cal-culated
rate
constant
from the pJ-ot
-ln ( [remaining
f or
disulf ide ] / [maximum reducib]-e
vs t j-me, for which slope :
disulfidel)
kapplDithiol ] .
c.
Reduct ion of
Dithiol
Using
a-Chymotrypsinogen
A
Samples of O-Chymotrypsinogen A (6. B mglml,, 0.2'7 mM)
(pH J I I mM in
in 50 mM sodium phosphate buffer
EDTA) at. room temperature
were reduced using 4. B mM
dithiol.
Under these reaction
conditions
a maxrmum
residue per Cr-Chymotrypsinogen
of 0.75 disulfide
A
(2) .
moLecuIe
was
reduced
The
ana'l vsis
for
reduction
of cr-Chymotrypsinogen
A was similar
to
that for trypsinogen.
D.
SDS-PAGE AnaLysis
ImmunogTobulin
by
Reduction
of
Dithiol
of
Samples of a murine i-mmunoglobul-in (IgGr, 6.3 mglml,)
(pH J , 0.5 mM in
in 50 mM sodium phosphate buffer
(BMS/ DMH, DTT; 4.8
EDTA) were reduced using dithiol
(25 LIL) of
m M ).
At several tj-me intervals,
aliquots
quenched
the
react ion
mixture
were
u s j,ng
(250 LIL of a 0.3 M iodoacetamide
iodoacetamide
'7,
solution
in 50 mM sodium phosphate buffer,
pH
1
mM in EDTA), and analyzed by 4-127 gradient SDS-PAGE
(2) .
under nonreducing conditions
263
R a p i d R e d u c t i o no f D i s u l f i d eB o n d s
Table
I.
Disulfide
DMH) 1
Comparisons
of
Bonds in Proteins
Protein
Rate Constants
for
Reduction
of
(DTT, BMS,
Reagents
Using Dithiol
kott
Reduction
keMs
knuH
korr
korr
Conditions
Trypsinogen
a-Chymotrypsinogen
Papain-S-SCH3
pH 7t
A
nIJ
vrr
'i
, ,
pH
'7
pH
1,
OoC
B M-1 min-1
1.1
2 eo..v
12 M-1 mi-n-1
9 M-1 min-1
,_t
- v
,'
) Ao^
)ao^
t
4 J
V
21OO M-I min-1
6.6
2.3
10
25
lRate
(k)
constants
are
rate
apparenc
constants
based
on
total
dithiol
concentration.
The calculations
rate
of
constants
are
in
described
Methods
The
rate
section.
constants
for
trypsinogen
and0-chymotrypsinogren
A are
from
reference
2.
IIT
Results
and
Discussion
Tabl-e I shows a comparj-son of the apparent
rate
constants
for the reduction
of disulfide
bonds in
proteins
using BMS, DMH and DTT.
BMS and DMH reduce
proteins
pH
1
the
disuffide
in
bonds
at
significantly
faster than does DTT.
The disulfide
bond in trypsinogen
is reduced
more rapidly
using BMS and DMH than using DTT by a
factor
of -1 (Tab]e I).
The rate of reduction
of
-202 faster
trypsinogen
by BMS is
than
by DMH
(Figure
1).
A maximum of 0.6 disulfide
residues
were reduced (i.e.
L.2 thiol
residues were formed)
per
t ryps inogen
molecule
under
react ion
these
conditions.
A selective
I1 9-203
cleavase
of
disulfide
bond in trypsinogen
has been reported
( 0.5
under
simil-ar
conditions
reduction
mM
of
dithioerythritol,
OoC, pH 8.5; Ref. 9).
The disulfide
bond in c-chymotrypsinogen
A is
reduced about 2.3-fold
faster
using BMS and DMH than
group
by DTT (Table I).
A maximum of 0.75 disulfide
per Cr-chymotrypsinogen A molecul-e was reduced under
reduction
the
The apparent
conditions.
rate
constant for the reduction
of disulfide
bond in
264
Ra.jeeva
Singh and George M. Whitesides
0.8
t
I
q)
bD
.--(t)
ar
-
0.6
F
h
L
0.4
F
a-
-s
F(
0.2
0.0
Time, min
Figure
1.
Reduction
Trypsinog'en
of
using
dithiols
IDTT
(5 mglml,, 0.2)BMS (I),
and DMH (A) l.
Trypsinog,en
(pH 7.0,
mM) in 50 mM sodium phosphate
buffer
1 mM in EDTA)
(0.5
was reduced
using
mM) at
dithiol
The curves
0oC.
plotted
are based on the values
of apparent.
rate
constants
I.
shown in Table
(O),
Cl-chymotrypsinogen A by DTT at 26"C is simi]-ar to
that for reduction
of trypsinogen
at OoC (Table I).
predicted
It is therefore
that the rate of cleavage
of disulfide
bond in Cr-chymotrypsinogen A would be
significanly
sl-ower than that for trypsinogen
at the
same temperature.
The L9I-220 disutfide
bond in Ct,chymotrypsinogen A is reported to be Iess accessible
than
the
anal-ogous
Il 9-203
in
disulf ide
bond
(9).
trypsinogen
The reactive
disulfide
bond in papain-S-SCH3 is
reduced especially
rapidly
by DMH (Figure 2, TabIe
I) .
The rates of reduction
of papain-SSCH3 using
DMH and BMS are
faster
DTT by
than
that
using
(Table I).
factors
of 25 and 10 respectively
The
group in papain has a.l-ow pKa (-4) and is
thiolfor
its
essential
The inactive
activity.
mixed
(papain-S-SCH3) is reactivated
disulfide
of papain
completely
within
5 min using small- concentrat ions
of DMH and BMS (Figure 2) .
265
R a o i d R e d u c t i o no f D i s u l f i d eB o n d s
€q)
100
+)
c!
L
q)
T
I
80
00
I
&
60
:a
*a
I
40
-ar
-
20
GI
A
-l
s
0
05101520
Time, min
Figure
2
Regeneration
of
activity
of
papain
from
papain-
DMH (a)].
s-scH3
IDTT
Papain-S-SCH3
rc.042 mg/mL, 1.8 Uvl in 50 mM sodium phosphate
dithiol
(pH 7, 2 mM in EDTA) at 23oC was reduced using
buffer
reaction
of
aliquots
intervals
(25 tlM).
time
At
severaland the activities
solution
were added to substrate
mixtures
ptotted
are based on the
The curves
were measured.
of papain
1'
shown in Table
rate
constants
values
of apparent
using
dithiols
(o),
BMs
(I),
and
bonds in immunoglobulin (IgGl) are
The disulfide
5
f
o
1
d
using DMH and BMS than using
f
a
s
ter
red.uced
Murine IgGr contains two heavy chains and
DTT (2) .
chains; the two heavy chains are l-inked to
two light
bonds, and each heavy
each other by two disulfide
bond
c
h
a
in by a disulfide
l
i
q
h
t
a
t
o
chain is linked
(10) .
SDS-PAGEanal-ysis of iodoacetamide-quenched
shows that
of IgGr and dithiols
mixtures
reaction
s
i
g
n
if icantly
t h e i m m u n o g r l - o b u l - i nm o l e c u l - e i s c l e a v e d
(
2
)
.
D
T
T
u
s
i
n
g
B
M
S
t
h
a
n
D
M
H
a
n
d
u
s
i
n
g
faster
Iv.
Conclus ions
bonds in proteins
Both BMS and DMH reduce disulfide
of -5-1 in
than does DTT by a factor
at pH 7 faster
266
R a j e e v aS i n g h a n d G e o r g eM . W h i t e s i d e s
nondenaturing conditions.
Although the typical
rate
enhancements expected
from using BMS and DMH over
-5
that
using
DTT
are
based
on
Branst.ed
correlations,
variations
are seen for some proteins:
the relatively
less accessibre disulfide
bond rn Gchymotrypsinogen A is reduced 2.3-fold
faster
using
BMS and DMH than using
DTT; the highly
reactive
disulfide
bond in papain-s-scH3
is reduced faster
using DMH than using DTT by a factor
of 25.
The
va]ues of equil-ibrium
constants
for the reduction
of
bis(2-hydroxyethyl)
(Eq 1) for BMS, DMH
disulfide
and DTT are
60 M, 2 M and 180 M respectively
(4,5111).
BMS is therefore
more reducing than DMH
and slightly
l-ess reducing
than DTT.
Al I these
(BMS, DMH, DTT) have significantly
dithiols
high
reduction potentials
and reduce noncvcric disul-fides
completely.
Although
both
BMS and
DMH reduce
disulfides
at similar
rates, we recommend the use of
BMS because it
is
commercially
available,
it
is
odorless and it has a hiqh reduction potential.
Re ference
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
s
Cleland, W. W. (1964). Biochemistry 3, 4BO-482.
S i n g h , R . , a n d W h i t e s i d e s , G . i \ , 1 .( 1 9 9 4 ) . B i o o r g . C h e m .
22, 109-115.
Singh, R., and Whitesides, G. M. (1993). In "Supplement
S: The Chemistry of Sulphur-Containing
Functional
Groups" (Patai, S., and Rappoport, 2., eds.) 633-659,
Wiley, London.
Lamoureux, G. V., and Whitesides, G. M. (1993). J. Org.
Chem. 58, 633-641.
Singh, R., and Whitesides, G. M. (1991). J. Org. Chem.
56, 2332-2337.
Lees, W. J., Singh, R., and Whitesides, G. M. (1991).
J. Org. Chem. 56, 7328-7331.
Singh, R., Bldttler,
W. A., and CoIIinson, A. R. (1993)
Anal. Biochem. 2I3, 49-56.
R i d d l e s , P . W . , B l a k e J - e y , R . L . t a n d Z e r n e r , B . ( 1 9 8 3 ).
I4ethods EnzymoJ. 91, 49-60.
Sondack, D. L. I and Light, A. (1971). J. Biol. Chem.
246, 1630-1637.
Edelman, G. M., and Gall, W. E. (1969). Annu. Rev.
Biochem. 38, 415-466.
Lees, W. J.,
58, 6 42-647 .
and whites j-des,
G. M.
(1993)
J.
Org.Chem.