Professional Documents
Culture Documents
THE JOUHNAL
OF Bmmo~cnr.
CHEMISTRY
244, No. 21, Issue of November
10, PD. 59285935,
Printed
in U.S.A.
1969
The Activation
of Papain and the Inhibition
Enzyme by Carbonyl
Reagents*
of the Active
(Received for publication,
IRA B. KLEIN
AND J.
F.
KIRSCH
SUMMARY
PROCEDURE
Materials
Radiochemicals-85S-Thiophenol,
chased from the Radiochemical
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-activated papain but neither inactivates nor binds to cysteine- or
borohydride-activated
papain in the presence of excess ac(b) The cysteine-activated
enzyme is also inhibited
tivator.
by phenylhydrazine
when the cysteine to papain ratio is low.
(c) This inhibition
in the presence of cyanide or low concentrations
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
Carboxamidomethylation
of the sulfhysite on the enzyme.
(e) Semidry1 group at the active site prevents this reaction.
carbazide
and hydroxylamine,
other reagents
having high
affinity for the carbonyl group, are much less effective inhibitors
of papain.
(f) Reactivation
of cyanide-activated,
peroxide-inactivated
papain by cysteine yields Z-iminothiazolidine-4-carboxylic
acid.
Model studies show that phenylhydrazine,
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
concomitant binding of the reagent.
These and other experiments show that the inhibition
of cyanide-activated
papain
by carbonyl reagents is not due to the affinity of these compounds for a carbonyl group on the enzyme, but rather to an
oxidative coupling of cyanide to the enzyme resulting
in the
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.
Issue of November
10, 1969
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 thiophenol, in which the method of Lowry et al. (18) was used.
A Packard Tri-Carb
model 3003 liquid scintillation
spectrometer was used to measure radioactivity.
Samples were
counted in 10 ml of a solution containing 5 g of 2,5-diphenyloxazole 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 phosphorothioate; Z-glycine p-NP, benzyloxycarbonylglycine
p-nitrophenyl
ester.
experiment, 0.5-ml samples were counted; in all other experiments, 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
(micrograms 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 described 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 activation 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 concentrations of papain ranging between 80 and 300 pM in the presence of
6 mM carbonyl reagents unless otherwise stated. Potassium cy-
5929
5930
Activation
TABLE
Extent of binding
Papain
or Bio-Gel
was activated
and then
P-2 as described
under
of Papain
to
of activators
and Inhibition
forms
various
eluted
through
Methods.
Amount
of papain4
Sephadex
of activator
Protein
G-10
bound
Thiol
per
reIessedb
?nolc/mole
K14CN
treatment
of
Inactive
papain
(7DB).
...
Carboxamidomethyl
papain
(7DB).
Cysteine
activated
papain
(activator
free) (7DB).
32P-P-thioate
Inactive
0.04
0.14
0.06
treatment
of
papain
(7DB).
0.09
0.18
0.01
0.04
A
35S-Cysteine
treatment
Papain
(IK-(cys))
of
.
0.07
-I
and
of 2 mM
[ Thiol
I/[
0.6
0.8
with
30-min
times.
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 Pthioate;
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).
RESULTS
PHI+ r
coo-L
-+
SCN
+ CYSTEINE
cyanide-activated
Worthington
papain was stable to rechromatography,
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 proteinbound 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 reillustrate a characteristic of papain which has been noted before
(3, 4, 6, 7, 25); that is, that the usual preparations of fully activated papain contain considerably less than 1 mole of thiol per
mole of protein, presumably indicating the presence of some irreversibly 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 activity 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).
(1)
The fact that cyanide also binds to a similar extent to carboxamidomethyland cysteine-activated
papain suggests that
the former explanation is more probable.
The cyanide bound to
anate
0.4
0.19
reaction
0.2
Issue of November
10, 1969
I. B. Klein
and J. F. Kirsch
5931
TABLE
Reaction
II
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 designated 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.
Esterase activity
.ctivator
Iresent
when
After
After
henyl.
1initial
phenylhyarctiva.
1hydrazine
razine
1:reatment
i adde b tion
Activator
--
After
reactivation
with
cystein@
Phenylhydrazine
1mund,per
pa:tn
1treatment
._
1soze/moze
None, native,
papain
Volume
Eluted
0.01
of
(mls)
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 determined using the optical density at 280 rn#; 0, thiol by the
method of Ellman (22) ; and A, radioactivity
of s2P and 3% as described under Methods.
2.
KCN
0.61
0.07: 1
0.64
0.076
0.61
0.061
ICl.75 0.043*
0.79
0.17
0.57
0.08
0.22
0.46
0.84
0.0476
0.57
0.01
0.42
0.066
0.049
I0.97
I0.91
Cysteine
I0.80
ID.89
Sodium borohydride
I0.97
ID.83
The chromatography
on Bio-Gel P-Z of 36S-cysteine- and 32P-Pthioate-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
The small thiol peak on the right of the graph correpapain.
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 unreacted further illustrates that the reaction of P-thioate with papain is a very efficient one at this dilute concentration
of activator, 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 reagent 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
(1After inactivation,
an aliquot of the mixture was diluted into
12 ml of the standard assay solution to a final enzyme concentration 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 investigated, phenylhydrazine
has been shown to be the most potent
inhibitor
of papain.
A particularly
puzzling aspect of the inactivation 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 explanation 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 inactivated 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 phenylhydrazine become bound to the protein.
The inactivation medi2 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.
None, carboxamidomethyl
derivative
papain
FIG.
0.12
inactive
5932
Activation
and Inhibition
of Papain
ated in the presence of cyanide was partly reversed by the addition 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 inactivation 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 suggest 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 cyanide 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
100
80
% Phenylhydrazine
TABLE
coupling
C,HaHNH,
SCN +
>
CeH,N&
NH*
/
-s-c
r
+ NH3
I$
(2)
\N-NH-C
6H 5
III
of WY-cyanide
The reactions
were carried
phosphate
buffer
containing
concentrations
varied
from
about
6 mM, cyanide,
1.6
activation
was done for 4
and hydrogen
peroxide
and
60 min, respectively.
CNCeH,NHNH,
or
H,Q
to activated
papain
Esterase activity
After
a, :tivation
After
phenylhydrazine
treatment
After
reactivation
CN bound per
nole of protein
before
reactivation
--
Carboxamidomethyl
papain
with
cyanide
and
ylhydrazine.
Cysteine-activated,
cysteine-free
paina
with
anide
and
hydrazine
Wphen0.06
paW-cyphenyl-
._1
-15
0.76
25
30
35
40
0.42
Volume
0.69
0.46
0.88
a Cysteine
was separated
from
papain
using
column
(1 X 15 cm), 0.01 M phosphate
buffer,
pH
a Bio-Gel
6.8, at 25.
i4C-Cyanide
activation
of papain
followed
by
Phenylhydrazine..
..
Hydrogen
peroxide.
.
0.69
0.69
0.070
20
0.11
0.05
P-2
(mls)
FIG. 3. Reversal
of phenylhydrazine
inactivation
by cysteine.
Phenylhydrazine-inhibited
cyanide-activated
papain
was prepared
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.
Oxidative
L-SH
Removed
Issue of November
10, 1969
TABLE
5933
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
inactivation
was completed
after 30 min.
Semicarbazide
and hydroxylamine were incubated
with the enzyme
for 2 hours unless otherwise indicated.
All reactions
were in 30 mM phosphate-30
mM
EDTA,
pH 6.8, at 25.
Esterase activity
Azeert tre&
After
activation
-1%~Semicarbazide
reacted
with cyanide-activated
papain
2 hours..
12 hours..
o.e3
0.61
0.93
0.71a
Hydroxylamine
reacted
W-cyanide-activated
pain.....................
0.17
0.19
3x10+
0.87
0.03
0.24
0.81
0.54
0.26
12 hours
after
semicarbazide
the enzyme
treatment.
was
The
TABLE
Cysteine
Papain
of papain
with
carbonyl
V
reagents
and
aldehyde
protection
Papain
(0.3 mM) was incubated
with the reagents
0.01 M phosphate
buffer,
pH 6.8, for the times shown,
activated
with 1.6 mM cyanide
for 4 hours.
Reagents added
indicated
in
after being
DUI%
tion 0
treatment
m.M
hrs
None
Phenylhydrazine
Phenylhydrazine
+
Benzaldehyde
Phenylhydrazine
+
Benzaldehyde
(M)
was
reacted
of
with
VI
against phenylhydrazine
papain
phenylhydrazine
inhibition
in the
presence
amounts
Esterase activity
Treatment
After
treatment
Recoverable
with more
activatora
None
0.82
0.90
Phenylhydrazine
3 mM for 1 hour
in dark
0.05
0.07
0.12
0.87
1.03
0.84
1.01
Esterase
activity
0.85
3.75
3.75
(added
together)
3.75
(added
sequentially)
Semicarbazide
+
Benzaldehyde
3.75
together)
0.31
0.08
0.91
0.59
0.58
was
added
hour
before
the
0.93
o Inactivation
mixture
(0.2 ml) was added to 12.17 ml of the
standard
assay solution
described
under
Methods
which
contained
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.
25.0
Benzaldehyde
mM)
1t
25.0
(3.75
0.04
25.0
3.75
(added
25.0
Semicarbazide
a Phenylhydrazine
mM benzaldehyde.
tion
6.8.
then
protection
Cysteine concentration
Concentrations of
reagents
3x10-2
Reagent
of
of cysteine.
The initial enzyme concentrawas 0.3 rnl\b in 2.5 mM phosphate
buffer,
2.5 mM EDTA
at pH
The protein
was incubated
25 min at 25 with cysteine
and
diluted
to 24 ,UM before treatment
with phenylhydrazine.
increasing
TABLE
of Carbonyl
with
pa-
a This
rate was determined
initially
activated,
but without
original
rate was 0.762.
Reaction
3x10+
Concentration
25
* 1 mole
of cvanide
I
W-Semicarbazide
reacted
with
peroxide-inactivated
cyanide-activated
papain.
W bound
per mole of
protein
carbonyl
reagent
5934
Activation
and Inhibition
coo-
SH
NH3+
+ N=X!%H~--C.i;
(3)
coo-
2-Iminothiazolidine4-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 reagents. 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 convincing, 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
There is no doubt howin acetic acid with denatured protein.
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 described above show that the mode of inhibition
of papain by
phenylhydrazine
is due to the ability of this compound to oxidize
of Papain
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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