-
THE JOUHNAL OF Bmmo~cnr. CHEMISTRY Vol. 244, No. 21, Issue of
November 10, PD. 59285935, 1969
Printed in U.S.A.
The Activation of Papain and the Inhibition of the Active Enzyme
by Carbonyl Reagents*
(Received for publication, May 29, 1969)
IRA B. KLEIN AND J. F. KIRSCH From the Department of
Biochemistry, University of California, Berkeley, California
94720
SUMMARY
The activation of papain with four different activators is not
accompanied by the binding of any of them to the protein. These
experiments, taken together with previously reported results, show
that the inactive form of papain prepared by the method of Kimmel
and Smith (J. Biol. Chem., 207, 575 (1954)) is a mixed disulfide
formed between the active site sulfhydryl group of the protein and
free cysteine.
The known inhibition of the activated enzyme by reagents having
affinity for carbonyl groups has been investigated in order to
determine whether an aldehyde residue which is intimately connected
with the activation process is present on the enzyme, or if the
observed inactivation of the protein by this class of reagents can
be accounted for in some other manner. The following relevant
observations were made. (a) Phenylhydrazine inactivates and binds
to cyanide-acti- vated papain but neither inactivates nor binds to
cysteine- or borohydride-activated papain in the presence of excess
ac- tivator. (b) The cysteine-activated enzyme is also inhibited by
phenylhydrazine when the cysteine to papain ratio is low. (c) This
inhibition in the presence of cyanide or low con- centrations of
cysteine is readily reversible with excess cysteine which also
releases the bound phenylhydrazine from the protein. (d) Treatment
of the enzyme with either phenylhydrazine or hydrogen peroxide in
the presence of WN- results in the binding of one cyanide group per
active site on the enzyme. Carboxamidomethylation of the sulfhy-
dry1 group at the active site prevents this reaction. (e) Semi-
carbazide and hydroxylamine, other reagents having high affinity
for the carbonyl group, are much less effective in- hibitors of
papain. (f) Reactivation of cyanide-activated, peroxide-inactivated
papain by cysteine yields Z-iminothia- zolidine-4-carboxylic acid.
Model studies show that phenyl- hydrazine, but not semicarbazide or
hydroxylamine, is capable of oxidizing cysteine to cystine during 1
hour of incubation with 30 mM reagent. Phenylhydrazine does not
bind to cyanate-inactivated papain. Activator-free papain is
irreversibly inhibited by phenylhydrazine, without con- comitant
binding of the reagent. These and other experi- ments show that the
inhibition of cyanide-activated papain by carbonyl reagents is not
due to the affinity of these com- pounds for a carbonyl group on
the enzyme, but rather to an oxidative coupling of cyanide to the
enzyme resulting in the
* This research was supported by National Institutes of Health
Grant GM12278 and National Science Foundation Grant GB4606.
conversion of the cysteine residue at the active site to @-
thiocyanatoalanine. Reactivation can be effected by excess
cysteine, whereby the cyanide moiety is transferred from the enzyme
to the free sulfhydryl group of the cysteine residue.
Papain, when isolated by the method of Kimmel and Smith (l), is
generally inactive and must be treated with either mer- captans
(l-5), cyanide (2-4), or other reducing agents (4) in order to
release the free active thiol and the concurrent prote- olytic or
esterase activity. The structure of the major fraction of inactive
papain has recently been demonstrated to be a mixed disulfide of
papain and cysteine (6, 7). The evidence in support of this model
is as follows. Activation of papain with K14CN results in the
release of thiol equivalent to the amount of cyclized
fl-thiocyanatoalanine released from the protein (6). This compound
is the expected product of the reaction of cyanide upon the
nonprotein sulfur atom of the mixed disulfide. Cys- teine can be
oxidatively bound to active papain, and the kinetics of
reactivation of this species are identical with those observed for
the activation of native papain (7). The activation is apparently
accomplished without a major conformational change as determined by
fluorescence, ultraviolet difference spec- troscopy, and circular
dichroism (8).
These recent findings are difficult to reconcile with other
phenomena associated with activation which have been de- scribed in
the literature. It has been reported for example that
phosphorothioate ion (PO&?) becomes reversibly bound to papain
during the course of activation (5). It has also been ar- gued,
from the sensitivity of papain to carbonyl reagents (%12), that an
aldehyde moiety is present on the protein and that the inactive
form is an hemithioacetal formed between the active site thiol
residue and this aldehyde (9, 12).
The experiments reported herein were designed to determine the
total extent of binding of activators during the course of
activation, and to elucidate the mechanism of the inactivation of
papain by carbonyl reagents.
EXPERIMENTAL PROCEDURE
Materials
Radiochemicals-85S-Thiophenol, 26.5 mCi per mmole, pur- chased
from the Radiochemical Centre (Amersham, England),
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Issue of November 10, 1969 I. B. Klein and J. F. Kirsch 5929
was diluted with redistilled unlabeled compound obtained from
Matheson, Coleman and Bell, East Rutherford, New Jersey, to a
specific activity of 173 cpm per mpmole. K14CN, about 6 mCi per
mmole, was obtained from New England Nuclear Corpora- tion and
diluted to a specific activity of 100 to 500 cpm per mpmole with
Mallinckrodt unlabeled KCN. Volk 35S-~- cysteine, 43.1 mCi per
mmole, was diluted with Calbiochem nn-cysteine HCl monohydrate to
243 cpm per mpmole. Ac- tivation with unlabeled cysteine was
performed using either Nutritional Biochemical Corporation
L-cysteine or the DL- cysteine HCl monohydrate from Calbiochem.
14C-Phenylhy- drazine HCl, 126 mCi per mmole from Tracerlab,
Richmond, California, was diluted with unlabeled compound obtained
from Eastman (White Label) to a specific activity of from 50 to 100
cpm per mFmole. Labeled P-thioatel was synthesized from 32PSC13
(Volk Radiochemical Company, Irvine, California) by the method of
ikerfeldt (13) to a final specific activity of 167 cpm per mpmole.
This preparation was used within a week of synthesis, and the
specific radioactivity was redetermined daily. 14C-Semicarbazide,
7.5 mCi per mmole, was purchased from Volk Radiochemical Company,
and diluted with unlabeled compound (Eastman White Label) to 50 cpm
per mpmole.
Other Reagents-Iodoacetamide was obtained from Calbio- them and
recrystallized from 1007, ethanol. Hydroxylamine HCl was purchased
from Fisher Scientific Company, Pittsburgh, Pennsylvania; hydrogen
peroxide as a 30% solution, Su- peroxol, from Merck; potassium
cyanate from Matheson, Coleman and Bell;
5,5-dithiobis(2-nitrobenzoic acid) from Aldrich and benzaldehyde
(White Label) from Eastman. NaBH4 was obtained from Metal Hydrides,
Inc., Beverly, Massachusetts. Bio-Gel P-2 was purchased from BioRad
Laboratories, and Sephadex G-10, G-25, and G-75 from Phar- macia
Fine Chemicals. Z-glycine p-NP was prepared by the method of
Bodanzky and du Vigneaud (14), m.p., 127.5128, literature 128.
Four papain preparations were used in these experiments:
Worthington Biochemicals, twice recrystallized enzyme, lot numbers
5629, 7DB, and 8CA, and enzyme purified in this laboratory from dry
papaya latex (Wallerstein Company, New York, New York) by the
method of Kimmel and Smith (l), as modified by Masuda (15). This is
referred to as IK(-cys). These Worthington Biochemical preparations
had similar maxi- mal velocities when assayed with Z-glycine p-NP,
while IK(-cys) was 60% as active. In the absence of added cysteine,
lot 5629 had an activity of 40% of the maximum obt.ainable (16),
while IK(-cys) had 25yo of its maximum rate that was obtained in
the presence of cysteine.
Methods
Enzyme concentrations were calculated from absorbance
measurements at 280 rnp using an extinction coefficient of 5.1 x
lo4 M- cm+ (17) except in the experiments with thio- phenol, in
which the method of Lowry et al. (18) was used.
A Packard Tri-Carb model 3003 liquid scintillation spec-
trometer was used to measure radioactivity. Samples were counted in
10 ml of a solution containing 5 g of 2,5-diphenyl- oxazole and 2 g
of 1,4-bis[2-(5-phenyloxazolyl)]benzene per liter of
toluene-ethanol mixture (19). In the ?S-thiophenol
1 The abbreviations used are: P-thioate, trisodium phosphoro-
thioate; Z-glycine p-NP, benzyloxycarbonylglycine p-nitrophenyl
ester.
experiment, 0.5-ml samples were counted; in all other experi-
ments, 0.2-ml samples were counted.
The enzyme was assayed essentially by the method previously
described (20). A catalytic rate was determined as follows.
Regardless of previous treatment, the enzyme was assayed in a
solution containing 20 mM phosphate buffer, pH 6.8, 1.0 mM EDTA,
and a final papain concentration of 0.3 to 0.7 PM. The reaction was
started by adding 0.2 ml of a 1.5 mM solution of Z-glycine p-NP in
redistilled acetonitrile to 2.8 ml of the above solution in a
cuvette maintained at 25 with a thermostat in the cell compartment
of a Zeiss PM& II spectrophotometer. The esterase activity was
calculated by dividing the initial change in optical density per
min at 400 rnp by the optical density at the end of the reaction.
This number was then divided by the protein concentration in
micrograms per ml and multiplied by 10 to give the reported
activity. A rate of 1.0 mini (micro- grams of enzyme per ml)- was
generally the highest attainable and was taken as 1OO70 or optimal
activity. For maximal recoverable activity after inactivation or
for the determination of maximal activity for a particular batch of
papain, a final concentration of 0.3 mM cysteine was used in the
standard assay solution as described above and allowed to react for
periods of up to 3 hours with the enzyme.
Activation of Papain Using Low Activator Concentrations-In one
activation experiment, the method of Soejima and Shimura (21) was
used with the following modifications. %-Thiophenol was used as an
activator instead of p-thiocresol, and the papain was not subjected
to prior inactivation with KI and Iz. The activation was performed
on Worthington lot 5629 in 40 mM citrate buffer, pH 5.0, at 25.
After gently shaking 2.5 ml of the 250 pM solution of papain with
2.5 ml of toluene containing 5 mM 35S-thiophenol, a 0.5-ml aliquot
of the aqueous phase was filtered through fine sintered glass and
then chromatographed on a column (1 x 8 cm) of Sephadex G-25 using
0.1 M phosphate buffer, pH 6.8, containing 1 mM EDTA at 25 under
nitrogen. The flow rate was about 1 ml per min. Fractions of 1.5 ml
were collected in serum capped tubes, assayed, and counted as de-
scribed above.
Papain was activated with K14CN as described previously (6)
Papain, which had been carboxamidomethylated or diluted due to
passage through a gel column, was at about 80 pM and was treated
with labeled 1.0 mM KCN. This cyanide treatment was generally
carried out for 4 hours. All experiments were done at pH 6.8 and 25
unless otherwise stated. In certain experiments, papain was made
activator-free after activation by elution through a Bio-Gel P-2
column (1 x 10 cm) in 0.01 M phosphate buffer. When activated by
3SS-cysteine, the papain was at 240 PM, and the labeled thiol at 2
InM; the activation mixture also contained 35 InM EDTA and 35 mM
phosphate buffer. Reaction time was 2 hours. The 3zP-P-thioate used
to activate papain was 0.17 mM as was the enzyme. Activation
proceeded for 1 hour in a 30 mM EDTA, 30 InM phosphate buffer, and
then the solution was chromatographed as described in Table I.
NaBH4 activa- tion was done with 180 mM NaBH4 in 0.67 mM EDTA and
25 mM phosphate buffer at pH 7 and 0 for 45 min, similar to the
procedure described by Glazer and Smith (4). Thiol titer was
determined by the method of Ellman (22).
Inactivation Conditions-Inactivation reactions were usually
performed for 1 hour at pH 6.8 and 25 in the dark at concentra-
tions of papain ranging between 80 and 300 pM in the presence of 6
mM carbonyl reagents unless otherwise stated. Potassium cy-
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5930 Activation and Inhibition of Papain Vol. 244, No. 21
TABLE I
Extent of binding of activators to various forms of papain4
Papain was activated and then eluted through Sephadex G-10 or
Bio-Gel P-2 as described under Methods.
K14CN treatment of Inactive papain (7DB). . . .
Carboxamidomethyl papain
(7DB). Cysteine activated papain (ac-
tivator free) (7DB).
32P-P-thioate treatment of Inactive papain (7DB).
35S-Cysteine treatment of Papain (IK-(cys)) .
Amount of activator bound per
Protein Thiol reIessedb
?nolc/mole
0.04
0.06
0.09
0.01
0.07 I -
0.14
0.18
0.04
0.19
D Lot 7DB was purchased from Worthington, while IK(-cys) was
isolated by the method of Kimmel and Smith (1) as modified by
Masuda (15).
b Based on thiol determination by the method of Ellman (22). The
thiol released per mole of protein in each experiment is given by
dividing the figure in Column 2 by that in Column 3.
anate and HzOz were used at concentrations of 2 mM with 30-min
reaction times.
RESULTS
On Binding oj Activators to Papain-The extent of binding of
a%-cysteine, K%N, and a2P-P-thioate to papain is shown in Table I.
After the incubation with the activator as described under Methods,
the mixtures were chromatographed on Sepha- dex G-10 or Bio-Gel P-2
to remove nonprotein-bound activator. It can be seen that neither
of the two thiol activators nor cyanide was bound in quantities
comparable with the enzyme thiol formed upon activation. The
binding of a%-thiophenol was also investigated on a sample of
papain that had some activity in the absence of activator (lot
5629), and the thiol was observed to bind to an extent of less than
0.1 mole per mole of protein thiol formed while fully activating
the enzyme.
It has been shown before (6), and here again, that only about
0.1 mole of C-cyanide was bound during cyanide-mediated ac-
tivation of papain per mole of protein thiol released. This could
be due to a nonspecific reaction of cyanide with one of the three
disulfide bridges on the papain molecule (23, 24) or to a wrong
side attack at the active site which would release free cysteine
from the inactive form and leave /3-thiocyanatoalanine at position
25 in the amino acid sequence as shown in Equation 1. This de-
rivative would presumably be inactive.
r PHI+ r -+ SCN + CYSTEINE L (1) coo-
The fact that cyanide also binds to a similar extent to car-
boxamidomethyl- and cysteine-activated papain suggests that the
former explanation is more probable. The cyanide bound to
1 A
0 0.2 0.4 0.6 0.8
[ Thiol I/[ Protein ]
FIG. 1. Enzymatic activity as a function of the number of
titratable -SH groups per mole of protein. All the experiments were
done on papain lot 7DB activated by either of three different
activators and at different ratios of activator to papain; v, 0.18
mM papain-O.18 mM P-thioate; A, 0.16 mM papain-O.8 mM P- thioate; l
, 0.3 mM papain-O.5 mM cysteine; n , 0.3 mM papain-3.0 mre CN-; 0,
no activator. The enzyme activity was measured as described under
Methods and the thiol titer was determined by the method of Ellman
(22).
cyanide-activated Worthington papain was stable to rechro-
matography, probably indicating that it is in covalent linkage with
the protein. The extent of binding of thiol activators to the
enzyme was likewise small compared with the amount of protein-
bound thiol released. Similar results using P-thioate and KCN were
obtained with another preparation of papain (lot 8CA).
The binding ratio is presented in two ways in the table to re-
illustrate a characteristic of papain which has been noted before
(3, 4, 6, 7, 25); that is, that the usual preparations of fully
acti- vated papain contain considerably less than 1 mole of thiol
per mole of protein, presumably indicating the presence of some ir-
reversibly inactivated papain. As shown in Fig. 1, the activity of
the enzyme is directly proportional to the titrable thiol on the
protein irrespective of the mode of activation. This result is
similar to the one obtained previously by Sanner and Pihl with no
added activator (2). At the maximum rate of the Z-glycine p-NP
assay of 1.0 mine1 (micrograms of enzyme per ml)+, this particular
batch of papain had 0.54 mole of SH per mole of protein based on
~280 = 51,000 (17). This rate of esterase ac- tivity is the highest
that could be obtained on several batches of Worthington papain,
and on papain isolated by the method of Kimmel and Smith (1) as
modified by Masuda (15) under many different activating
conditions.
In some of the experiments it was necessary to use low ratios of
activator to protein so that all of the potential protein-bound
thiol was not released. Since the amount of nonspecific binding of
the activators was fairly constant, a small thiol titer resulted in
a higher molar ratio of bound activator to thiol released as is
seen by the difference between the amount of activator bound per
mole of protein and per mole of thiol (Table I).
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Volume Eluted (mls)
FIG. 2. Bio-Gel P-2 chromatography in 10 mM phosphate buffer, pH
6.8, at 25. A, 2 mM 3%-cysteine-activated 0.24 mM papain in 35 mM
phosphate, 35 mM EDTA, pH 6.8, for 2 hours. B, 0.170 rnM
32P-thioate activation of 0.170 mM papain in 30 mM phosphate
buffer, 30 mM EDTA for 1 hour. 0, protein was de- termined using
the optical density at 280 rn#; 0, thiol by the method of Ellman
(22) ; and A, radioactivity of s2P and 3% as de- scribed under
Methods.
The chromatography on Bio-Gel P-Z of 36S-cysteine- and 32P-P-
thioate-activated papain is shown in Fig. 2. The elution profiles
show that a small amount of radioactivity comes out with the void
volumes of the columns and was taken to be protein bound. These
elution profiles are similar to the one previously shown for
W-cyanide-activated papain (6), except that Bio-Gel P-2 was used
here instead of Sephadex G-10. Fig. 2B illustrates how small an
amount of P-thioate is bound to papain, but, more interestingly,
shows that a new 32P-containing compound is formed as a result of
the reaction of P-thioate with inactive papain. The small thiol
peak on the right of the graph corre- sponds to the elution of
P-thioate when chromatographed alone. By assuming an analogous mode
of activation to that by cyanide, and because oxidized P-thioate,
ostensibly the disulfide (03PSSP03) comes out at a different point
than either the large azP-containing peak, the protein peak, or the
thiol peak, this new product is considered to be a mixed disulfide
of cysteine and P-thioate. The small amount of P-thioate that
remains unre- acted further illustrates that the reaction of
P-thioate with pa- pain is a very efficient one at this dilute
concentration of acti- vator, as has been reported previously by
Neumann, Shinitzky, and Smith (5).
From the fact that cyanide-activated papain is particularly
sensitive to inhibition by carbonyl reagents (10, ll), Morihara et
al. (9,12) have postulated that the inactive form of the enzyme has
a structure in which the active thiol is bound as an internal
hemithioacetal. The reactions of papain with this type of re- agent
were, therefore, investigated in order to attempt to find a
satisfactory explanation for this phenomenon.
The Reaction of Papain with Carbonyl Reagents-Of the
reagents
TABLE II
Reaction of phenylhydrazine with papain
Papain concentrations between 80 and 300 pM were activated with
4- to lo-fold molar excesses of cysteine or cyanide, at 25, or with
a 450 molar excess of NaBH4 at pH 6.8 to 7.0 at 0. A 30- to
loo-fold excess of phenylhydrazine was employed to inactivate the
activated papain at 25 for 1 hour at pH 6.8. Where desig- nated as
activator-free, the activated papain was eluted through a Bio-Gel
P-2 column (1 X 15 cm), 0.01 M phosphate buffer, pH 6.8, before
treatment with phenylhydrazine.
Activator
None, native, inactive papain
None, carboxamido- methyl derivative of papain
KCN
Cysteine
Sodium borohydride
.ctiva- tor
resent when henyl.
hy- razine i adde
- ! I-
1
b a
--
I
I
I
I
I
I
I
-
Esterase activity
After initial rctiva. tion
0.97 0.91
0.80 D.89 Cl.75
0.97 D.83
1 1
._
-
After phenyl-
hydrazine :reatment
0.066 0.049
0.61 0.061 0.043*
0.84 0.0476
-
- After reacti- vation with cys-
tein@
0.61 0.07:
0.79 0.17 0.57
0.57
- I
1
1
1
1
-
Phenyl- hydrazine mund,per pa:tn
treatment
soze/moze 0.12
0.01
0.64 0.076
0.08 0.22 0.46
0.01 0.42
(1 After inactivation, an aliquot of the mixture was diluted
into 12 ml of the standard assay solution to a final enzyme
concentra- tion of 0.3 to 0.7 PM. Two portions of this diluted
solution were assayed immediately, and the remaining two portions
were treated with 0.3 mM cysteine for 3 hours and then assayed to
determine recoverable activity as described under Methods.
* A lo-fold molar excess of cyanide over protein was added with
the phenylhydrazine.
having affinity for carbonyl groups which have been investi-
gated, phenylhydrazine has been shown to be the most potent
inhibitor of papain. A particularly puzzling aspect of the in-
activation of papain by phenylhydrazine is that the inhibition is
more pronounced when papain is activated by cyanide than when it is
activated by cysteine (9, 12). The experiments reported in Table II
confirm this observation, and provide a plausible ex- planation for
it. Papain activated either by KCN, cysteine, or NaBH4 was
inactivated after the activator had been removed by gel filtration
before treatment with phenylhydrazine. Under these conditions, very
little radioactive phenylhydrazine was bound to the protein as a
result of the inactivation process. When cyanide was used as the
activator and was not removed prior to the addition of
phenylhydrazine, papain was also in- activated by the
phenylhydrazine. In this case about 1 mole of phenylhydrazine was
bound per mole of active enzyme.2 If either cysteine or NaBH4 was
present during the phenylhydrazine treatment, there was no
appreciable inactivation, nor did phenyl- hydrazine become bound to
the protein. The inactivation medi-
2 According to Fig. 1, there is about 0.5 mole of free thiol,
and, according to Table III, about 0.5 mole of phenylhydrazine
bound.
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5932 Activation and Inhibition of Papain Vol. 244, No. 2I
ated in the presence of cyanide was partly reversed by the addi-
tion of excess cysteine, and this reactivation was accompanied by
the displacement of phenylhydrazine from the enzyme (Fig. 3). The
inactivation caused by phenylhydrazine in the absence of activator
cannot be reversed by cysteine. The requirement for cyanide for
binding of phenylhydrazine and reversible inactiva- tion was
explored further by the experiments shown in Table III. The results
demonstrate that, in addition to the presence of cyanide being
required for the binding of r4C-phenylhydrazine to papain (Table
II), either phenylhydrazine or hydrogen peroxide will couple
14C-cyanide to the enzyme. These experiments sug- gest that the
mechanism of inactivation of papain in the presence of cyanide by
these reagents is not due to a reaction of a carbonyl group on the
enzyme, but rather to an oxidative coupling of cya- nide to the
active sulfhydryl group of the enzyme converting the essential
cysteine residue at position 25 in the primary sequence
0.0 w 0 20 40 60 80 100
% Phenylhydrazine Removed
FIG. 3. Reversal of phenylhydrazine inactivation by cysteine.
Phenylhydrazine-inhibited cyanide-activated papain was pre- pared
as described in Table II. The inactivated papain was
chromatographed on Bio-Gel P-2. Two fractions containing 40 and 30
PM papain were treated with 0.3 mM cysteine for 15 hours, and 30 mM
cysteine for 24 hours, respectively, at pH 6.8 and 25 in 10 mM
phosphate buffer. Rechromatography on Bio-Gel P-2 was done to
determine activity recovered and the remaining bound
phenylhydrazine as described under Methods.
L- -SH CN- CeH,NHNH, > or H,Q
SCN + C,HaHNH,
- CeH,N& + NH3
I$ (2) r NH* / -s-c \N-NH-C H 6 5 TABLE III
Oxidative coupling of WY-cyanide to activated papain
The reactions were carried out at pH 6.8 and 25 in 10 or 30 mM
phosphate buffer containing 1 mM or 10 mM EDTA. Papain
concentrations varied from 80 to 400 PM. Phenylhydrazine was about
6 mM, cyanide, 1.6 mM, and peroxide, 2 mu. Cyanide activation was
done for 4 hours; cysteine activation, 45 min; and hydrogen
peroxide and phenylhydrazine inactivation 30 and 60 min,
respectively. -
I I Esterase activity CN bound per nole of protein
before reactivation
After phenyl-
hydrazine treatment
After reactiva-
tion After
:tivation a,
--
-
Carboxamidomethyl papain with W- cyanide and phen-
ylhydrazine.
Cysteine-activated, cysteine-free pa- paina with W-cy- anide and
phenyl- hydrazine
i4C-Cyanide activation of papain followed
by Phenylhydrazine.. . . Hydrogen peroxide. .
0.06
._1 30 35 40 -15 20 25
0.76
0.69 0.69
0.070
0.11 0.05 0.69
0.42 Volume (mls)
FIG. 4. The oxidative binding of cyanide to the active site of
papain. Sephadex G-75 chromatography in 10 mu phosphate buffer, pH
6.8, of cyanide-activated papain (see Methods). A, half the
activation mixture was inactivated with 20 mM phenyl- hydrazine. B,
the other half was treated with 50 mM iodoaceta- mide which
completely abolished the activity, and then followed by 20 mM
phenylhydrazine. Protein (0) was determined using the optical
density at 280 rnp, and radioactivity (A) as described under
Methods.
0.46 0.88
a Cysteine was separated from papain using a Bio-Gel P-2 column
(1 X 15 cm), 0.01 M phosphate buffer, pH 6.8, at 25.
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Issue of November 10, 1969 I. B. Klein and J. F. Kirsch 5933
TABLE IV
Hydroxylamine and semicarbazide reactions with cyanide-activated
papain
Papain (0.4 mM) was activated with 1.6 mM cyanide for 4 hours at
25 in 0.01 M phosphate buffer, and then incubated with 6 mM
carbonyl reagent or 3 mM Hz02. The hydrogen peroxide inacti- vation
was completed after 30 min. Semicarbazide and hydroxyl- amine were
incubated with the enzyme for 2 hours unless other- wise indicated.
All reactions were in 30 mM phosphate-30 mM EDTA, pH 6.8, at
25.
Esterase activity
After activation
Azeert tre&
carbonyl reagent
W bound per mole of
protein --
-
1%~Semicarbazide reacted with cyanide-activated papain
2 hours.. 12 hours..
W-Semicarbazide reacted with peroxide-inactivated
cyanide-activated papain.
Hydroxylamine reacted with W-cyanide-activated pa-
pain.....................
0.93 o.e3 0.17 0.71a 0.61 0.19
0.87
0.81
0.03
0.54
0.24
0.26
3x10+ 3x10+ 3x10-2
Concentration of Carbonyl Reagent (M)
FIG. 5. The oxidation of cysteine by carbonyl reagents. Cys-
teine (24 PM) was treated with semicarbazide (A), phenylhydra- zine
(O), or hydroxylamine (0) for 1 hour at 25, pH 7.0 (10 mM
phosphate, 1 mM EDTA). Thiol was determined by the method of Ellman
(22). Cysteine which was not treated with carbonyl reagents showed
less than 2% loss of thiol under these conditions. -
a This rate was determined 12 hours after the enzyme was
initially activated, but without semicarbazide treatment. The
original rate was 0.762.
TABLE VI
Cysteine protection of papain against phenylhydrazine inhibition
Papain was reacted with phenylhydrazine in the presence of
increasing amounts of cysteine. The initial enzyme concentra-
tion was 0.3 rnl\b in 2.5 mM phosphate buffer, 2.5 mM EDTA at pH
6.8. The protein was incubated 25 min at 25 with cysteine and then
diluted to 24 ,UM before treatment with phenylhydrazine.
TABLE V
Reaction of papain with carbonyl reagents and aldehyde
protection Papain (0.3 mM) was incubated with the reagents
indicated in
0.01 M phosphate buffer, pH 6.8, for the times shown, after
being activated with 1.6 mM cyanide for 4 hours.
-
-
Esterase activity
Cysteine concentration Treatment -
Recoverable with more activatora Concentra- tions of
reagents
DUI% tion 0 treat- ment
After treatment
0.82
0.05
0.12 0.87 1.03
Esterase activity Reagents added
None
Phenylhydrazine Phenylhydrazine
+ Benzaldehyde (added together)
Phenylhydrazine +
Benzaldehyde (added sequentially)
Semicarbazide
Semicarbazide
+ Benzaldehyde (added together)
Benzaldehyde
None
Phenylhydrazine 3 mM for 1 hour in dark
m.M
3.75 3.75
25.0
3.75
25.0
3.75
3.75
25.0
25.0
hrs
0.85
1 0.04
1 0.31
1
1t 0.08
1 0.91
1 0.59
1 0.58
hour before the 25 a Phenylhydrazine (3.75 mM) was added mM
benzaldehyde.
0.90
0.07
0.84 1.01 0.93
- o Inactivation mixture (0.2 ml) was added to 12.17 ml of
the
standard assay solution described under Methods which con-
tained a final concentration of 35 mM phosphate, pH 6.8, 1 rnM
EDTA, and 0.5 pM papain. One-half of this was assayed, and the
remainder was made 0.3 mM in cysteine, incubated at 25 for 3 hours,
and then assayed to determine the recoverable activity.
(23, 24) to P-thiocyanatoalanine. These reactions a.re sum-
marized in Equation 2.
The proposed structure of the adduct produced by the addition of
phenylhydrazine to the thiocyanate group is based on the
stoichiometrv of the binding of atmroximatelv 1 mole of cvanide u
II * I
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5934 Activation and Inhibition of Papain Vol. 244, No. 21
per mole of phenylhydrazine, and is supported by analogous model
reactions (see Discussion). The location of the bound cyanide at
the active site cysteine is strongly suggested by the fact that
alkylation at this position completely prevents the bind- ing of
cyanide caused by phenylhydrazine (Fig. 4).
The oxidation hypothesis was further tested by the experi- ments
reported in Table IV, wherein it is shown that neither one of two
other well known carbonyl reagents, semicarbazide or hydroxylamine,
significantly inhibit cyanide-activated papain under these
conditions, nor is 14C-semicarbazide bound to the enzyme to any
appreciable extent. It is shown below that these compounds are poor
oxidizing agents as well. Hydroxylamine, moreover, does not cause
as much 14C-cyanide to bind to papain as does phenylhydrazine or
hydrogen peroxide.
Benzaldehyde has been reported to provide partial protection
against the phenylhydrazine-induced inactivation of the enzyme
(11). This result is confirmed by some of the experiments shown in
Table V, where it is observed in addition, that benzaldehyde itself
causes some inactivation of the enzyme. The protection by
benzaldehyde need not, however, reflect a competition be- tween
this aldehyde and a similar group on the enzyme, but only the
complexation of the phenylhydrazine presumably through the
formation of the benzaldehyde phenylhydrazone. The inacti- vation
caused by the benzaldehyde itself was not further in- vestigated,
but may be due to the formation of a hemithioacetal between the
active site thiol group and the added aldehyde. It should be
further noted that benzaldehyde was only able to moderate the
phenylhydrazine-induced inhibition when it was added with the
phenylhydrazine; benzaldehyde added after treatment with
phenylhydrazine was totally ineffective in re- versing the
inhibition of the enzyme.
The oxidative inactivation hypothesis was further tested by the
model experiments shown in Fig. 5 where phenylhydrazine, but
neither semicarbazide nor hydroxylamine, is shown to com- pletely
oxidize cysteine at a concentration of 0.03 M. This observation,
along with the experiments reported in Table VI, provides a
plausible explanation for the fact that phenylhydra- zine does not
inactivate cysteine-activated papain in the pres- ence of excess
cysteine; i.e. sufficient cysteine remains after treatment with
phenylhydrazine to either prevent oxidation of the enzyme or to
reactivate the oxidized papain. Papain itself is oxidized in 1 hour
at a phenylhydrazine concentration of one- tenth the amount
necessary to oxidize cysteine.
Sluyterman (26) has shown that cyanate ion inactivates papain by
carbamylation at the active site thiol. The possibility that the
inactivation of the enzyme promoted by oxidizing agents in the
presence of cyanide is due to carbamylation by cyanate formed by
the oxidation of cyanide was excluded by the following two
observations. (a) The extent of r4C-phenylhydrazine bind- ing to
carbamylated papain prepared from activator-free papain, which was
inactivated by 20-fold molar excess of potassium cyanate was only
0.15 mole per mole of protein as opposed to an average of 0.65 mole
per mole of protein when cyanide itself was present; and (b)
incubation of the enzyme with cysteine after it was inactivated
with hydrogen peroxide in the presence of r4C- cyanide resulted in
the formation of 2.iminothiazolidine-4- carboxylic acid, presumably
by the mechanism shown in Equa- tion 3.
\ coo-
r NH3+ SH + N=X!%H~--C.i; (3) L \ coo- \
2-Iminothiazolidine- 4-carboxylic acid
This process is the reverse of the mechanism of activation of
papain described previously (6). This product could not have arisen
from X-carbamylated papain, and its appearance proves that the
cysteine used in the reactivation experiments must attack the
thiocyanato moiety at the carbon rather than the sul. fur atom,
since 2-iminothiazolidine-4-carboxylic acid, rather than CN-, was
released.
DISCUSSION
The radioactively labeled mercaptans, YS-thiophenol, 3%-
cysteine, and 3P-P-thioate, along with Kr4CN did not bind to the
protein in sufficient quantity to account for the amount of active
thiol released on activation of the enzyme by these re- agents.
These results do not support models that require that the
activation process consists of a noncatalytic cleavage of a simple
intramolecular bond of the active thiol of papain.
Although the evidence discussed above for an inactive form of
papain in which there is a mixed disulfide formed between a
sulfhydryl group on the enzyme and cysteine (6, 7) is convinc- ing,
there are experiments suggesting other inactive forms of papain
that must be explained. Some of these have led to proposals in
which the sulfhydryl group which, after activation, becomes the
catalytic nucleophile of the enzyme, is actually bound
intramolecularly to some group on the polypeptide chain (5, 9, 12).
It has been reported in one instance that the binding of the
activator, P-thioate, was proportional to the increase in the
catalytic activity of the enzyme; the conclusion being based upon
observed changes in fluorescence of the protein upon activation,
and on the binding of the activator to the enzyme as shown by
Sephadex chromatography in 0.1 M acetic acid. In contrast to the
former result, Bare1 and Glazer (8) have concluded that the
activation of papain has little effect on the ultraviolet circular
dichroism or the extent of perturbation of the enzymes aromatic
chromophores. The experiments we have just presented show that no
activator, including P-thioate, binds to papain under the
activation and assay conditions described in this paper.
We have no definite explanation for the differences between our
results and those in the literature (5) with respect to the
apparent binding of P-thioate. This reagent is known to attack
dithio bridges in proteins (27) and such a reaction might have
occurred in acetic acid with denatured protein. There is no doubt
how- ever that papain activity is not dependent upon the continued
presence of the activator (Table I) (2, 6, 8, 21).
Although the inhibition of papain by carbonyl reagents has been
interpreted in terms of a reaction of this reagent with a critical
aldehyde moiety on the enzyme, the experiments de- scribed above
show that the mode of inhibition of papain by phenylhydrazine is
due to the ability of this compound to oxidize
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Issue of November 10, 1969 I. B. Klein and J. F. Kirsch 5935
nanain rather than to the formation of an enzyme-bound phenyl-
4. GLAZER, A. N., AND SMITH, E. L., J. Biol. Chem., MO, 201
hydrazone. This conclusion is further supported by the obser-
vation that carbonyl reagents such as semicarbazide and hy-
droxylamine, which do not have the ability to oxidize cysteine at
low concentrations (Fig. 5), are not effective inhibitors of the
enzyme (Table IV).
5. (1965)
NEUMANN, H., SHINITZKY, M., AND SMITH, R. A., Biochemistry, 6,
1421 (1967).
6.
7.
The proposed structure of the phenylhydrazine-thiocyanato papain
adduct shown in Equation 2 is, to a certain extent, speculative,
but it is supported by the fact that phenylhydrazine will not bind
to the protein unless cyanide is also present, and that these two
compounds are bound in approximately equal stoichiometry (Tables II
and III). The structure is, moreover, chemically reasonable since
the thiocyanato carbon atom is known to form adducts under mild
conditions with nitrogen nucleophiles as occurs in the cyclizations
of P-thiocyanatoalanine to form 2-iminothiazolidine-4-carboxylic
acid (28) and o-amino- thiocyanatobenzene to form
2-aminobenaothiazole (29). The fact that there is more cyanide
bound when peroxide rather than phenylhydrazine inactivates
CN--activated papain may be be- cause a similar mechanism is
operating in which a second mole of cyanide attacks the carbon atom
of the initially formed thio- cyanato group; a reaction which is,
to a certain extent, analogous with the dimerization of HCN
(30).
KLEIN, I. B., AND KIRSCH, J. F., Biochem. Biophys. Res. Com-
mun., 34, 575 (1969).
SLUYTERMAN, L. A. AL, Biochem. Biophys. Acta, 139, 430
(1967).
8. BAREL, A. O., AND GLAZER, A. N., J. Biol. Chem., 244, 268
(1969).
9. M~RIH~RA, K., J. Biochem. (Tokyo), 62, 250 (1967). 10.
BERGMANN. M.. AND Ross. W. F.. J. Biol. Chem.. 111. 659
11. (1935).
,
BERGMANN, M., AND Ross, W. F., J. Biol. Chem., 114, 717
(1936).
12. MORIHARA, K., MONNA, K., AND AKABORI, S., Proc. Jap.
13. 14.
Acad., 41, 828 (1965). ARERFELDT. S.. Acta Chem. Stand.. 14.
1980 (1960). BODANZKY, M.; AND DU VIGNEAUD V.,.Biochem. Prep., 9,
110
(1962). 15. MASUDA, T., J. Biochem. (Tokyo), 46, 1489 (1959).
16. HENRY, A. C., AND KIRSCH, J. F., Biochemistry, 6, 3536 (1967).
17. GLAZER, A. N., AND SMITH, E. L., J. Biol. Chem., 236, 2948
(1961). 18. LOWRY. 0. H., ROSEBROUGH, N. J., FARR, A. L., AND
RANDALL,
The treatment of activator free papain with oxidizing agents
leads to an inactive form of the enzyme that cannot be reacti-
vated with cysteine. The nature of this species is unknown, but it
presumably arises through the conversion of the active site
cysteine residue to a sulfinic or a sulfonic acid. In the presence
of activating nucleophiles, the intermediate papain sulfenium ion
is trapped (Equation 2), preventing further irreversible oxi-
dation.
19. 20. 21.
R. J.; J. Biol. Chem., 193; 265 (1951). HALL. T. C.. AND
COCKING. E. C.. Biochem. J.. 96. 626 (1965). KIRS~H, J. f., AND
IGELSTR~M, M:, Biochemist&, 5; 783 (1966). SOEJIMA, M., AND
SHIMURA, K., J. Biochem. (Tokyo), 49, 260
(1961). 22. 23.
24.
25.
Acknowledgments-We are grateful to Dr. W. Dixon Riley for the
gift of Tracerlab 14C-phenylhydrazine, and we wish to thank Mrs.
Patricia Hinkle for preparing the carboxamidomethylated derivative
of papain.
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Ira B. Klein and J. F. Kirsch
Reagentsof the Active Enzyme by Carbonyl The Activation of
Papain and the InhibitionMACROMOLECULES:CHEMISTRY AND METABOLISM
OF
1969, 244:5928-5935.J. Biol. Chem.
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