The Effect of Urea and Guanidine Hydrochloride on Activity

and Optical Rotation of Crystalline Papain*

ROBERT L. HILL, HERBERT C. SCHWARTZ,? AND EMIL L. SMITH

From the Laboratory for the Study of Hereditary and Metabolic Disorders, and the Departments of Biological Chemistry
and Medicine, University of Utah Collegeof Medicine, Salt Lake City, Utah

(Received for publication, September 29, 1958)

The catalytic activity of several proteolytic enzymes is altered The Rudolph Precision Polarimeter No. 80 with a sodium
by high concentrations of urea and guanidine salts (2-4). These lamp was used. Observed rotations were estimated from the
studies, in conjunction with certain physical data, indicate that average of 10 consecutive readings and are expressed as specific
hydrogen bonding in the secondary structure of these enzymes rotations calculated in the usual manner.
is essential for maintenance of their catalytic function. Because
of the increasing amount of information that is available con- RESULTS

cerning the structure of papain (5, 6), and its mechanism of

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Effect of Urea and Guanidine Hydrochlorideon Activity-Figs.
action (7-lo), it was of interest to determine whether this en- 1 and 2 show the effect of urea and guanidine hydrochloride on
zyme is affected by concentrations of urea or guanidine hydro- the activity of papain.1 In these experiments solutions of
chloride which alter secondary, structural hydrogen bonding.
mercuripapain contained the final concentration of reagent that
This paper presents the results of such studies. is indicated in the figures. Aliquots of each of these solutions
In contrast to the work of Lineweaver and Schwimmer (11) were assayed in reaction mixtures containing the same urea or
who reported no inactivation of papain by 9 M urea, our results
guanidine hydrochloride concentration. Other aliquots were
demonstrate that even lower levels of urea cause considerable
assayed in aqueous solutions in which the urea or guanidine
inactivation. Although urea reversibly inactivates papain,
hydrochloride concentration was diluted to 1 per cent that of
high levels of guanidine hydrochloride were found to cause an
the original solution. The estimated activities are plotted in
irreversible inactivation, accompanied by significant unfolding
Figs. 1 and 2 at the final concentration of either urea or guanidin-
of the enzyme.
ium ion.
EXPERIMENTAL It is apparent that both agents cause considerable inactiva-
tion. The specific activity decreased sharply between 0.1 and
Materials and Methods-Crystalline mercuripapain was pre- 2 M urea, with almost complete loss of activity at 6 M concentra-
pared by a modification of the procedure of Kimmel and Smith tion and complete inactivation at 8 M concentration. The
(12). In all experiments in which mercuripapain was exposed activity of papain after exposure to concentrations of urea up
to urea or guanidine hydrochloride, the aqueous diluent was to 3 M and then dilution, was that expected on the basis of the
either 0.05 M or 0.1 M sodium acetate buffer at pH 5.1, unless small amount of urea present, an indication that the inactiva-
noted otherwise. pH measurements were made with a Beckman tion was completely reversible under these conditions. How-
model G or a Cambridge model R pH meter, and no attempt ever, the activities, after exposure to concentrations of urea
was made to correct the observed pH for effects of urea or guani- greater than 3 M, were less than that expected from the final
dine hydrochloride. Recrystallized guanidine hydrochloride, urea concentration. Complete reactivation would give a curve
prepared from reagent grade guanidine carbonate, and recrystal- superimposable on the urea inactivation curve. This apparent
lized reagent grade urea were used in all experiments. partial reversibility has also been demonstrated by the fact that
Assays of enzyme activity were performed as previously
papain after exposure to 6 M urea and subsequent removal of
described (12) with 0.05 M cr-benzoyl-L-argininamide as substrate
urea by dialysis, shows only 50 to 60 per cent of its original
at pH 5.1, in the presence of 0.005 M cysteine and 0.001 M ethyl-
specific activity.
enediaminetetraacetate. Specific activity (C,) was calculated
With guanidine hydrochloride the specific activity decreased
from k1 (first order rate constant calculated in decimal log-
sharply between 0.1 and 3 M showing almost complete inactiva-
arithms) per mg. of protein N per ml. of reaction mixture. Pro-
tein concentration was estimated by a turbidimetric method tion at 4 M. The activity of papain after exposure to concentra-
(13). In the experiments performed in the presence of urea tions of guanidine hydrochloride up to 0.5 M and then dilution,
or guanidinium ion, there was no interference with estimation was that expected from the small amount of reagent present.
of enzyme activity. Partial reversibility occurred at concentrations up to 3 M, whereas
papain exposed to concentrations greater than 4 M was irrevers-
* This investigation was aided by research grants from the
National Institutes of Health, United States Public Health Serv-
ice. A preliminary report of this work has been presented (1). 1 In these experiments and the ones to follow no differences
t Postdoctoral Fellow, National Heart Institute, United States could be found between the results obtained with crystalline
Public Health Service. mercuripapain or crystalline papain.

572
March 1959 R. L. Hill, H. C. Xchwartx, and E. L. Smith

80-

after
Dilution

20

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t I
I I I
2 I 0 -I
-LOG MOLAR UREA CONC. -LOG MOLAR GUANIDINE CONC.
FIG. 1. Effect of urea on papain activity. Urea was added to FIG. 2. Effect of guanidine hydrochloride on papain activity.
solutions of mercuripapain to give the final concentrations shown. These experiments were performed as described in Fig. 1 for the
The concentration of enzyme varied from 0.38 to 1.0 per cent. urea inactivation studies. 0, Activity in guanidine hydrochlo-
After at least 30 minutes at 25”, activity was estimated at 40” ride; 0, activity after dilution of guanidine hydrochloride to 1
with or-beneoyl-r-argininamide as substrate at pH 5.1. Assays per cent of original level.
were performed at the concentration of urea used for denatura-
tion, 0, and after dilution to 1 per cent of the original level of
urea, 0. I I I 1 ’
ibly inactivated. Such irreversibly inactivated papain was very No guanidine
insoluble after removal of the denaturing agent.
In order to determine whether hydrolysis of a protein sub- 0.8
strate by papain is altered by denaturing agents in the same
manner as the hydrolysis of the synthetic substrate, a-benzoyl-
L-argininamide, the following experiment was performed. To
solutions of human serum albumin buffered at pH 5.1 with 0.05
M acetate buffer containing 0.005 M dimercaptopropanol, enough
guanidine hydrochloride was added to give the final concentra-
tions shown in Fig. 3. Mercuripapain was then a.dded and the
extent of hydrolysis estimated by the increase in ninhydrin
color at 570 rnp (14) on aliquots removed at intervals after
starting the reaction. The results are shown in Fig. 3. Al-
though guanidine hydrochloride caused a definite decrease in
the rate of hydrolysis, the percentage inhibition is not as great
as that found for comparable concentrations of guanidine hy- 60 120
drochloride with the synthetic substrate (Fig. 2). Despite the
fact that papain was not exposed to guanidine hydrochloride TIMER, MINUTES
until the beginning of the reaction, the rate of inactivation of FIG. 3. Rate of hydrolysis of human serum albumin by papain
papain by guanidine hydrochloride, which is described below, in guanidine hydrochloride. The extent of hydrolysis was esti-
mated by the increase in ninhydrin color at 570 rnp on aliquots
should be sufficiently rapid to produce a similar degree of in- removed at intervals after starting the reaction. Although guani-
activation to that seen with the synthetic substrate. It is thus dine hydrochloride gives some color, each measurement was cor-
apparent that the albumin protected the papain to some extent rected by an appropriate blank.
from denaturation2 However, no hydrolysis occurred after 60
minutes in the mixture containing 4 M guanidine hydrochlo-
2 In a single experiment performed in the same manner as those
ride.
shown in Fig. 3, it was observed that mercuripapain, treated with
4 M guanidine hydrochloride for 30 minutes, subsequently gave Time Dependence of Guanidine Hydrochloride and lirea In-
no hydrolysis of albumin. activation-The time dependence of the effects of increasing urea
574 E$ect of Urea and Guanidine Hydrochloride on Papain Vol. 234, No. 3

I I I I I I ’ TABLE I
’ ’ ’ ’ ’ ’ ’
GUANIDINE UREA of urea on specific rotation
E$ect of papain
Solutions of mercuripapain, 0.4 to 1.0 per cent, were prepared
in buffers at the indicated pH either in the absence or presence of
6 M urea. The observed rotations did not change over a 4%hour
period.
0
PH [a!? in water (a]~0 in urea

4.3 (-) 65.3” (-) 77.6”

I I I I I I 1 I I I 5.7 (-) 66.7 (-) 68.2
12 24 36 I2 24 36 8.0 * (-) 62.3
TIMENMINUTES 10.0 * (-) 68.4
FIG. 4. Time dependence of guanidine hydrochloride and urea
inactivation. In each instance the proteolytic coefficient (C,) * Insufficient light transmitted for precise estimation.
was calculated as a function of time at the concentration of de-
naturing agent shown. Results are expressed as per cent of the
activity of the papain preparation in water. 0 IF -65”
and guanidine hydrochloride concentrations on the specific
activity of papain is shown in Fig. 4. In these experiments
mercuripapain was added to solutions of a-benzoyl-n-arginin-
amide in 0.05 M acetate buffer at pH 5.2 containing 0.005 M 20 l-

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dimercaptopropanol and the denaturing agent at the concentra-
tions indicated in Fig. 4. Aliquots of these mixtures were then
used to determine the extent of hydrolysis of oc-benzoyl-n-
argininamide at various times. Maximal inhibition of papain )-
at all three concentrations of guanidine hydrochloride occurs
after 12 minutes. In addition, urea exerts its maximal inhibi- .80”
tion within 20 to 30 minutes. Other experiments have shown
that the activity of papain in 1 M urea does not change over 72 I-
hours, whereas the activity of papain in 6 M urea decreases over
72 hours to 4 per cent of the original activity. No differences
in the time dependence of denaturation could be found over the
range from pH 4.3 to 10. 8C)-
It is noteworthy that the experiments shown in Figs. 1 and 2
were performed after 30 minutes of exposure to the denaturing
agent. Consequently, all of these studies represent maximal
inhibition by urea or guanidine hydrochloride.
IOC)- -9!Y
Efect of Urea and Guanidine Hydrochloride on Optical Rotation
of Papain-Although papain activity is extremely sensitive to U.b u.9
urea, little effect could be found on the optical rotation. Table LOG MOLAR GUANIDINE CONC.
I lists the average specific rotations of papain solutions in the FIG. 5. Specific rotation [ol]:” of papain in guanidine hydro-
absence and presence of 6 M urea at different pH values. An chloride solutions. Solutions of mercuripapain, 0.4 to 0.6 per
increase in the levorotation was found at pH 4.3, however, cent, were prepared in 0.06 M acetate buffer at pH 5.2 containing
the indicated concentration of denaturing agent.
little change was observed at pH 5.7. In view of the low solu-
bility of papain at pH 8 to 10 and the fact that insufficient light
is transmitted it was impossible to obtain precise readings for DISCUSSION
the aqueous solutions. The specific rotations at these pH values From the results presented it is evident that papain is inacti-
in 6 M urea suggest that little or no change in rotation has oc- vated by high concentrations of urea or guanidine hydrochloride.
curred, and we cannot judge whether the slightly lower rotation These findings are not in accord with those of Lineweaver and
found at pH 8.0 in 6 M urea is significant. Although these Schwimmer (11) who indicated that papain is fully active in 9
results were obtained at the D-line of sodium, different results M urea. Although some irreversible inactivation should have
might be obtained at other wave lengths, It is important to occurred it appears that these authors evaluated the activity of
note that no increase in rotation occurred even after 48 hours urea solutions of papain under assay conditions which involved
of exposure to urea. dilution. This was the case in a number of the early experi-
Fig. 5 shows the change in optical rotation of papain as a ments concerned with the stability of enzymes in urea and has
function of guanidine hydrochloride concentration. Unlike its been emphasized previously (2, 15). However, protein sub-
behavior in urea, papain shows considerable unfolding in guani- strates were used by the above workers and, indeed, we have
dine hydrochloride as judged by the increasing levorotation. found that human serum albumin tends to stabilize papain to a
Although no effect is found below 2 M guanidine hydrochloride, considerable extent against inactivation by guanidine hydro-
the maximal increase in levorotation occurs at 4 M concentra- chloride. This situation also obtains with other proteolytic
tion. It is also at this latter concentration that papain is ir- enzymes, in particular, trypsin, chymotrypsin, and carboxy-
reversibly inactivated (Fig. 2). peptidase (16, 17).
March 1959 R. L. Hill, H. C. Schwartz, and E. L. Smith 575

An attempt to obtain such evidence has been made by deter-

mining the nature of the inhibition produced by low concentra-
tions of urea. The results are presented in Fig. 6 as a Line-
6
weaver-Burk plot.3 It is apparent that this inhibition is of the
so called “uncompetitive type” (18). Because the slope of the
lines in the presence and absence of inhibitor is the same and
numerically a function of [I + K,(l)]/koe, it can be concluded
that koe is unchanged by inhibitor. Since it has been shown
that K, = t&,/k1 for papain (6), and since the data of Fig. 6
indicate that koe is unchanged by the presence of inhibitor, the
primary effect of urea must be on kr, i.e. the step involving the
interaction of enzyme and substrate. An alternative explana-
tion for the effect of low concentrations of urea and guanidine
salts might then be found in the kr step which represents the
formation of the enzyme-substrate complex, or more specifically,
the formation of an acyl-thiol-enzyme complex (8). Urea or
guanidine salts could then be interfering either directly with
the formation of the intermediary thiol ester or with side chain
.04 .05 .06 .07 interaction of the guanidinium portion of the substrate 01-
6) benzoyl-L-argininamide and the enzyme. The structural simi-
FIG. 6. A plot of initial substrate concentration (S) divided by larity between the guanidinium group of cr-benzoyl-arginin-
velocity V (per cent hydrolysis per minute) versus (S) for the amide and both urea and guanidine hydrochloride might lend

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hydrolysis of 0.05 M cu-benzoyl-L-argininamide at 40” and pH 5.2 support to interference with the side chain interaction in the
in the presence of three different concentrations of urea.
kl step. An alternative plot of the data of Fig. 6 where log
V
In studies with other proteolytic enzymes, it has been ob- (p - 1) is plotted against log I yields a line with a slope of
VI
served that alteration of the secondary structure, as judged by
4.2 f 0.2; this has been interpreted to mean that 4 molecules
changes in optical rotation or viscosity, parallels inactivation
of inhibitor interact with the enzyme.
of the enzyme (2, 3). This effect has been interpreted as in-
Regardless of the explanation of the effects of urea and guani-
dicating that hydrogen bonds of the secondary structure stabilize
dine salts in inactivating papain, it is apparent that papain does
the active site of the enzyme. Rupture of these hydrogen bonds
differ from other proteolytic enzymes in that it is reversibly
thus leads to destruction of the active site. In the case of inactivated by low concentrations of these reagents which do
papain, however, significant loss of enzymic activity occurs at
not produce measurable unfolding of the molecule by the cri-
concentrations of urea or guanidinium ion which do not affect
terion of optical rotation. However, it is also apparent that
its secondary structure as judged by the fact that no measurable
papain is similar to many other enzymes in that high concentra-
change in optical rotation can be detected. The first change
tions of the above reagents produce irreversible inactivation
in rotation is found between 1 and 2 M guanidine hydrochloride
accompanied by unfolding of the enzyme. Thus a folded struc-
(Fig. 5) whereas 50 to 60 per cent of the activity of papain is ture is essential for the catalytic activity of papain in spite of
destroyed at these concentrations (Fig. 2). One explanation
the fact that a considerable portion of the primary peptide
for these results is in terms of the proposed structure of the
structure is dispensable for papain activity (19).
catalytic site of papain. Kinetic studies have demonstrated
that a thiol group of a cysteine residue and a carboxyl group SUMMARY
function in the active site (7-9), and it has been suggested that
these form the active site by an interaction which forms a “high 1. Papain is reversibly inactivated by low concentrations of
energy” thiol ester or hydrogen bond stabilized by the folding urea and guanidine hydrochloride. This inactivation is not
energy of the protein (10). A possible explanation for the effect accompanied by a change in secondary structure as judged by
of urea or guanidine salts is that these agents can interfere with optical rotation experiments.
the interaction of the thiol and carboxyl groups of the active 2. Although little change is found in the specific rotation of
site at concentrations which do not disrupt hydrogen bonds in papain in 6 M urea, it is almost completely although reversibly
the enzyme as judged by the lack of effect on the rotation. inactivated at this concentration. Guanidine hydrochloride,
However, additional evidence is needed before this interpreta- 5 M, irreversibly inactivates papain, producing a maximally
tion can be accepted. unfolded molecule as judged by optical rotation measurements.

REFERENCES
1. HILL, R. L., SHIELDR, G. S., SCHWARTZ, H. C., AND SMITH, GER (Editor), Symposium on protein structure, John Wiley
E. L., Federation Proc., 17, 242 (1958). and Sons, Inc., 1958, p. 182.
2. HARRIS, J. I., Nature, 177, 471 (1956). 3 The kinetics of hydrolysis of ol-benzoyl-n-argininamide by
3. NEURATH, H., RUPLEY, J.A., AND DREYER, W.J., Arch.Bio- papain were treated in terms of the conventional formulation
&em. Biophys., 66, 243 (1956).
h ko
4. PERLMANN, G. E., Arch. Biochem. Biophys., 66, 210 (1956). Ei-S - ES + E+P
5. KIMMEL, J. R., AND SMITH, E. L., Advances in Enzymol., 19, -icy--
267 (1957). For papain, K, = ko/kl (7). Kr is the inhibition constant for
6. SMITH, E.L., HILL, R.L., AND KIMMEL, J.R.,inA. NEUBER- urea and (I) is the concentration of urea.
576 E$ect of Urea and Guanidine Hydrochloride on Papain Vol. 234, No. 3

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(1958) line enzymes, 2nd Edition, Columbia University Press, New
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 The Effect of Urea and Guanidine Hydrochloride on Activity and Optical
Rotation of Crystalline Papain
Robert L. Hill, Herbert C. Schwartz and Emil L. Smith
J. Biol. Chem. 1959, 234:572-576.

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