PLANT PROTEASES

I. ACTIVATION-INHIBITION REACTIONS

BY DAVID M. GREENBERG AND THEODORE WINNICK

(From the Division of Biochemistry, University of California Medical School,
Berkeley)

(Received for publication, May 13, 1940)

The recent work of Balls and Lineweaver (1) with crystalline

papain supports the view that one or more sulfhydryl groups are
essential to the activity of this enzyme. These investigators have

Downloaded from http://www.jbc.org/ by guest on January 21, 2020

adopted the term papainases to characterize the various plant
proteolytic enzymes which resemble papain in their reversible
activation and inhibition reactions.
It is commonly stated that the properties of bromelin of pine-
apple are the same as those of papain, although no thorough
study of the former enzyme has been made. In the present paper,
the nature of the active groups of bromelin is investigated with the
aid of reagents that have come into recent use for this purpose.
In addition, the activation-inhibition reactions of two other pro-
teases are described. One of these enzymes is from the horse-
nettle, Solanum elaeagnijolium, whose milk-clotting action was
first studied by Bodansky (2). The name solanain is suggested
for this enzyme.1 The other protease, hitherto unreported, is
from the latex of the milkweed, Asclepias mexicana. We have
recently reported on the properties of the protease from a different
milkweed, Asclepias speciosa (3), and in this paper these enzymes
are distinguished by the names asclepain m and asclepain s.
Certain experiments with papain and asclepain s, designed to
test the sulfhydryl theory, are also described. It will be shown
that bromelin, asclepain m, and asclepain s are papainases, but
that solanain gives none of the reactions suggestive of the presence
of an active sulfhydryl, and accordingly, the last enzyme is not
a papainase.
1 It is suggested by Dr. H. Lineweaver and by us that the ending “ain”
be used to form the generic names of new proteases from plant sources.
761
762 Plant Proteases. I
Isolation of Proteases
Bromelin--To obtain this enzyme, 3 liters of juice from fresh
pineapple fruit2 were filtered with the aid of super-eel, and ad-
justed to pH 6 with ammonia. Then solid (NH&S04 was added
to the point of saturation. The resulting precipitate of crude
enzyme was centrifuged down, collected on a Buchner filter, and
washed with 0.6 saturated (NHJ2S04. The precipitate was
then redissolved in a liter of 0.02 M NaCN (pH S), and the solution
again made 0.6 saturated with (NH4)2S04. The precipitate, col-
lected as before, was drained as dry as possible, and redissolved

Downloaded from http://www.jbc.org/ by guest on January 21, 2020

in 600 ml. of 0.02 M NaCN. Then the enzyme was precipitated
by the addition of 3 volumes of acetone. It was centrifuged
down, collected on a Buchner filter, washed with acetone followed
by ether, and finally dried in a vacuum desiccator. The almost
colorless, dry product that resulted weighed 5 gm.
Asclepain m-10 ml. of latex from Asclepias mexicana were
extracted with 10 ml. of 0.05 M NaCN (pH 7), and the filtrate
saturated with solid (NH4)2S04. The resulting precipitate was
collected on a filter, washed with 0.7 saturat,ed (NH&S04, drained
free of liquid, and then dried in a vacuum desiccator. The light
yellow product weighed about 100 mg.
Solanain-This enzyme was prepared from the fruit of Solanum
elaeagnijolium.4 Bodansky’s isolation procedure (2) was modified
as follows :
The fresh fruit was ground and extracted with dilute phosphat’e
buffer (pH 7.5), and the extract centrifuged free of solids. The
dark green solution was made up to about 0.7 saturation with
solid (NH&S04. The resulting precipitate of crude enzyme was
centrifuged down, collected on a Buchner filter, washed with 0.7
saturated (NHJ2S04, and drained dry. The precipitate was

2 Pineapple fruit was kindly sent to us by Dr. J. L. Collins of the Pine-

apple Producers Experiment Station of the University of Hawaii.
3 This milkweed, which was found growing wild near Pinole, California,
differs greatly from Asclepias speciosa in size an8 structure. It is a small
weed, usually growing to less than a foot in height, and it yields but a drop
or two of latex per plant.
4 Collections of the fruit were kindly sent to us by Dr. R. Chandler of
t,he Botany Department, and Dr. W. H. Brown of the Zoology Department,
of the Universit,y of Arizona.
 D. M. Greenberg and T. Winnick
readily dissolved in water, and the enzyme reprecipitated by
adding 4 volumes of acetone. The precipitate was washed with
acetone and drained free of liquid. It was again dissolved in
water, precipitated by 0.7 saturation with (NH&S04, and col-
lected on a filter. Finally, the enzyme preparation was precipi-
tated twice more from aqueous solution by 4 volumes of acetone,
washed with acetone and ether, and dried in a vacuum desiccator.
About 0.8 gm. of white product was the yield per 100 gm. of fresh
fruit.
Asclepain s and Papain-These enzymes were prepared as
previously described (3).

Downloaded from http://www.jbc.org/ by guest on January 21, 2020

Methods
The activating or inhibiting effect of different reagents on the
proteolytic activity of the enzymes was measured by the same
technique used in previous studies with asclepain s. The enzyme
solutions were treated with the specified reagents for an hour at
room temperature in the cases of asclepain m and bromelin, and
for about 12 hours in the experiments with solanain. Then the
proteolytic activity of the treated solutions was measured on
2 per cent Van Slyke casein at 40” by the Northrop and Kunitz
non-protein nitrogen method (4). In the measurements with
asclepain m and bromelin, the casein substrate, buffered at pH
7.5, was digested for 30 minutes. For solanain the substrate was
buffered at pH 8.5, and the digestion time was 60 minutes. The
original enzyme solutions contained 1.5 mg. of asclepain m, and
3.0 mg. of bromelin or solanain, per ml.
The degree of activation or inhibition is expressed in each case
as the ratio of the activity of the treated enzyme to that of the
untreated enzyme solution. The activity values, expressed as
milliequivalents of non-protein nitrogen produced in 6 ml. of
digestion mixture in a definite time, are recorded for the untreated
enzymes, but are omitted for all the treated enzyme solutions, and
only the ratios are given. Enzyme solutions, when treated with
maleic acid, always stood at least 12 hours before further treat-
ment.
In certain special experiments, reported in Table III and else-
where in the text, proteolytic activity was measured by Anson’s
hemoglobin method (5).
764 Plant Proteases. I

DISCUSSION

Asclepain m and Bromelin-The results of the activation-

inhibition measurements with asclepain m and bromelin are given
in Tables I and II. The behavior of these enzymes, in most
TABLE I
Activalion-Znhibition of Asclepain m
The untreated enzyme solution, diluted with 2 volumes of water, pro-
duced 0.172 to 0.178 milliequivalent of non-protein nitrogen in 6 ml. of
digestion mixture.

Downloaded from http://www.jbc.org/ by guest on January 21, 2020

Reagent added to enzyme solution iatio of activity to
that of untreated
enzyme
Inhibitor Activator
-
None 0.05 M cysteine 2.98
“ 0.1 “ NaCN 2.78
“ 0.1 ” H,S 2.66
0.005 M Hz02 None 0.02
Same 0.05 M cysteine 2.70
0.001 M K3Fe(CN)6 None 0.02
Same 0.1 M NaCN 2.74
0.0005 M I, None 0.06
Same 0.1 M NaCN 0.15
0.01 M iodoacetic acid None 0.01
Same 0.05 M cysteine 0.00
0.03 M maleic acid None 0.08
Same 0.1 M NaCN 2.65
5 mg. CUZO None 0.04
Same 0.1 M H,S 0.08
0.001 N Ag+ or Hg++ None 0.00-0.02
1 X 1lP N Ag+ I‘ 0.20
3 x 10-6 I( i( ,‘ 0.52
1 x 10-6 (i ci ‘I 0.82
0.01 N Ag+ 0.1 M HzS 0.04
Same 0.1 “ NaCN 1.46
0.01 N Hg++ 0 1 “ H,S 0.48
o:l “ I‘
0.001 “ Ag+ 2.00
Same 0.1 “ NaCN 2.12

respects, resembles the effects that have been reported for papain
and asclepain s.
It has been pointed out by Anson (6) and by Hellerman (7)
that sulfhydryl groups with different degrees of reactivity may
be present in different native proteins, and sometimes in a single
 D. M. Greenberg and T. Winnick 765

protein. Balls and Lineweaver (8) likewise conclude that the

-SH of native crystalline papain is able to react with certain

TABLE II
Activation-Inhibition of Bromelin
The untreated enzyme solution, diluted with 2 volumes of water, pro-
duced 0.223 to 0.255 milliequivalent of non-protein nitrogen in 6 ml. of
digestion mixture.
Reagent added to eneyme solution
II ietio
that
of activity
of untreated
to

Inhibitor ACtiW&tOr CIlZYl3W

Downloaded from http://www.jbc.org/ by guest on January 21, 2020

None 0.06 M cysteine 2.28
“ 0.03 “ “ 2.04-2.16
,‘ 0.1 “ NaCN 2.15
I‘ 0.1 “ HkJ 1.18-1.25
‘C 0.5 ” NaaS 1.10
0.003 M Hz02 None 0.04
Same 0.1 M NaCN 0.84
0.01 M KaFe(CN)e* None 0.60
Same 0.1 M NaCN 1.90
0.001 M KMnO* None 0.16
Same 0.1 M NaCN 1.18
0.001 M 12 None 0.00
Same 0.03 M cysteine 0.26
0.02 M iodoacetic acid None 0.01
Same 0.1 M NaCN 0.00
0.03 M maleic acid None 1.08
Same 0.1 M NaCN 2.18
5 mg. Cu10 None 0.08
Same 0.1 M H,S 0.18
0.01 N Ag+ or Hg++ None 0.00-0.02
1 X 1OF N Ag+ I‘ 0.22
2x IO-6 (6 I( “ 0.62
0.67 X 10-e N Ag+ ‘I 0.82
0.01 N Ag+ 0.1 M H# 0.01-0.04
Same 0.5 “ NazS 0.50
0.1 “ NaCN 1.76-1.90
0.001 N Ag+ or Hg++ 0.1 “ HzS 1.26-1.28
0.001 “ “ 0.1 “ NaCN 2.12
* This reagent was allowed to act for 12 hours.

reagents but not with others. If it is assumed that reversibly

oxidized thiol groups in enzymes can likewise have different re-
 Plant Proteases. I
activities, one has a further basis for interpreting activation-
inhibition differences. The responses of the enzymes depend not
only upon the oxidation-reduction potentials of the reagents, but
upon the particular conditions that determine the reaction
rates (9).
Activation by Reducing Agents-In Table I it is seen that ascle-
pain m is activated to approximately the same degree by different
reducing agents if sufficiently high concentrations are employed.
This resembles the finding of Balls and Lineweaver (1) with
crystalline papain. It seems likely that cysteine, cyanide, and
H$ all cause the same chemical change. To account for this on

Downloaded from http://www.jbc.org/ by guest on January 21, 2020

the basis of the formation of thiol groups in the enzymes, it must
be assumed that CN- is oxidized to CNO- and H&S to S.
In the case of bromelin (Table II), cysteine and cyanide activate
to about the same degree, while H&S and NazS produce a much
lower degree of activation. Rather than postulate a different
reaction product in the latter case, as was tentatively done by
Hellerman and Perkins (10) for crude papain, it may be simply
that the oxidized sulfhydryl groups of bromelin are not readily
reducible by sulfides.
Fruton and Bergmann (11) found that papain, activated with
cysteine or cyanide, becomes completely inactive to synthetic
peptides when precipitated by isopropyl alcohol, and that the
enzyme precipitate recovers full activity when again treated with
the activators. This led them to favor the older view that these
activators form dissociable compounds with papain, and therefore
function as coenzymes rather than as reducing agents.
Experiments like those of Fruton and Bergmann were carried
out by us on papain and asclepain s. The details are, briefly,
as follows :
Preparations of asclepain s and papain were dissolved in 0.05
M neutral cysteine or cyanide solutions. The enzymes were pre-
cipitated with 4 volumes of ethyl or isopropyl alcohol, collected
on Buchner filters, and washed with absolute alcohol. From the
precipitates, solutions were made up that contained equal con-
centrations (0.2 mg. per ml.) of each enzyme in both pure water
and in 0.05 M HCN (pH 7). The proteolytic activity of these
solutions was then measured by Anson’s hemoglobin method,
with 10 minute digestions at 30”.
 D. M. Greenberg and T. Winnick 767
The results in Table III show that asclepain s, activated with
cysteine or HCN, loses only 10 per cent of its activity in the above
precipitation procedure. Activated papain loses about half its
activity when precipitated by ethyl or isopropyl alcohol. When
the papain precipitate was strongly aerated, there was a large
irreversible loss of activity.
In the opinion of the authors, the reversible losses in activity
are probably produced by mild oxidation of the enzymes by
oxygen, owing to the presence of catalyzing impurities, and not
to dissociation of an enzyme-activator compound. The energetic
aeration of the papain precipitate probably oxidized the active

Downloaded from http://www.jbc.org/ by guest on January 21, 2020

TABLE III
Effect oJ Alcohol Precipitation on Activity of Asclepain s and Papain

i Proteolytic activity of
redissolved enzyme
(tyrosine in 6 ml. digestion
Alcohol used to ppt. mixture)
Enzyme Initial activator ensyme
NOTI- Enzyme
activated treated with
IXGSyIIle HCN

m.eq. x 108 m.eq. x 108

Asclepain s Cysteine Ethyl 6.7 7.3
HCN ‘L 6.4 7.2
Papain Cysteine ‘I 3.0 6.6
“ Isopropyl 3.2 7.1
“ Ethyl, ppt. aerated 0.45 1.3
30 min. on filter

sulfhydryl groups beyond the reversible stage. It is significant

that highly purified papain is stable toward oxygen, and can be
salted-out of 70 per cent alcohol by lithium sulfate, and subse-
quently recrystallized from water without loss of activity (1).
This evidence opposes the view that alcohol dissociates a papain-
activator complex.
Reversible Inactivation by Oxidants-When asclepain m is com-
pletely inactivated by dilute Hz02 or ferricyanide, 90 to 100 per
cent of the full activity can be subsequently restored by the addi-
tion of excess cyanide or cysteine. These effects strongly suggest
an oxidation-reduction process. An explanation in terms of the
dissociable complex theory seems less plausible.
768 Plant Proteases. I

When bromelin is inactivated by dilute Hz02 or KMnO+ subse-

quent treatment with cyanide does not produce full activation.
This suggests that part of the sulfur is oxidized beyond the re-
versible stage. The partial inactivation obtained with ferricya-
nide (acting for a much longer time) and the subsequent
reactivation closely resemble the effects obtained by Hellerman
and Perkins with impure papain (10). Apparently, ferricyanide
is unable to oxidize all of the sulfhydryl, but, like Hz02 and KMn04,
can oxidize part of it past the reversible stage. It may be noted
in this connection that the “free” sulfhydryl groups of native egg

Downloaded from http://www.jbc.org/ by guest on January 21, 2020

albumin do not react at all with ferricyanide (6), even at pH 9.6.
Ketene-The irreversible inactivations found with iodine suggest
that aromatic groups may be iodinated in these reactions. In
connection with this possibility, it was of interest to test the action
of ketene on papain.6 This reagent inactivates pepsin presumably
by acetylating the phenolic hydroxyl groups in the enzyme (12).
The course of the inactivation of papain by ketene was found to
resemble that of pepsin. Ketene, acting on a 0.1 per cent solution
of papain (activated with HCN) at O”, and buffered at pH 5.5,
caused a 20 per cent loss in activity in 5 minutes, and a 70 per cent
loss in 1 hour.
Since it seemed possible that ketene might acetylate the -SH
groups of papain, this was guarded against in another experiment
by mildly oxidizing the enzyme with dilute Hz02 before subjecting
it to acetylation. In this experiment the papain lost 55 per cent
of its potential activity in 5 minutes, and 95 per cent in 1 hour,
upon treatment with ketene.
These results suggest that ketene may react with groups in
papain other than sulfhydryl, which are also essential for proteo-
lytic activity. The slow rate of the reaction makes it seem
unlikely that these are primary amino groups. Balls and Line-
weaver (1) likewise believe that the NH2 groups of papain are
unrelated to the enzyme activity. Accordingly, in line with the
deductions for pepsin, it seems possible that phenolic groups, in
addition to -SH, are essential for the proteolytic activity of
papain.
Iodoacetic Acid-This reagent, which reacts vigorously with
6 We thank Dr. C. H. Li of the Institute of Experimental Biology of the
University of California for the use of his ketene generator.
 D. M. Greenberg arid T. Winnick 769
-SH compounds, completely inactivates asclepain m and bro-
melin. None of the activity is restored by adding an excess of
activator. Balls and Lineweaver (8) have shown that the inhibi-
tion of papain activity is produced by 1 molecular equivalent of
iodoacetate, and have detected hydriodic acid as a product of
the reaction, so that the reaction mechanism seems fairly well
established.
According to Maschmann (13), the inactivation of papain by
iodoacetate is reversed by precipitation of the enzyme with alco-
hol. This experiment was repeated by us, using 0.02 M iodo-
acetic acid (pH 7) completely to inactivate a 0.3 per cent papain

Downloaded from http://www.jbc.org/ by guest on January 21, 2020

solution. Following alcohol precipitation, we found no recovery
in the proteolytic activity of the enzyme.
Cuprous O&e-This substance, which reversibly inactivates
papain at pH 5 (lo), inactivates asclepain m and bromelin irre-
versibly at pH 7. The nature of the reaction here is not known.
Maleic Acid-Morgan and Friedmann (14) found that the
incubation of papain with maleic acid at 37” and at pH 4.7 pro-
duced 70 per cent inhibition of the enzyme after 16 hours. By
analogy with the reactions of thiol compounds, these workers
conclude that maleic acid probably forms an addition compound
with reduced papain. No attempt was made to reverse the
inactivation.
We have obtained 90 per cent inhibition of papain with maleic
acid at pH 7.0 by incubation for 5 hours at 37”. This inactivation
was about 70 per cent reversed by the subsequent addition of
excess cyanide.6 In Table I it is seen that asclepain m is com-
pletely inhibited by maleic acid, and is fully reactivated by
cyanide. Asclepain s behaves in the same manner (data not
recorded).
A plausible explanation of the reversal in these cases is that
maleic acid inactivates by oxidizing the enzymes, and that it is
itself reduced to the rather stable substance, succinic acid; then
cyanide reactivates by reducing the oxidized forms of the enzymes.
6 The same conditions were employed as in measurements with asclepain
m and bromelin, except that the hemoglobin method was used to measure
proteolytic activity. Following the inactivation by 0.01 M maleic acid, the
papain solution (containing 0.6 mg. of enzyme per ml.) was activated to
68 per cent of the full activity value for the cyanide-treated enzyme.
770 Plant Proteases. I
The -SH of bromelin apparently does not react readily with
maleic acid, as its activity is not appreciably altered by this
reagent. When excess cyanide is also added to the solution, the
enzyme becomes fully activated.
Heavy Metal Ions-Hellerman (9) explains the inactivation of
papain by heavy metal ions as being due to the formation of
mercaptides. Activators, such as HCN and HSS, remove the
metal and liberate the -SH groups of the enzyme.
When asclepain m and bromclin are inactivated by adding 0.001
N Ag+ or Hg++ to the enzyme solutions, it is possible, by the
subsequent addition of excess sulfide or cyanide, to activate

Downloaded from http://www.jbc.org/ by guest on January 21, 2020

asclepain m to 75 per cent, and bromelin to 100 per cent, of the
full latent activity. But when 0.01 N metal ions are added,
excess sulfide produces 0, or only slight activation, while excess
cyanide reactivates asclepain m about 50 per cent, and bromelin
about 85 per cent. This depressed reactivation, which results
when relatively concentrated solutions of Ag+ or Hg++ are used,
is most marked with asclepain s. The activation of this enzyme
by 0.001 N Ag+ is completely reversed by 0.1 M H2S (data not
recorded). But if 0.01 N Ag+ is used, HZS restores none of the
proteolytic activity (3). The reason for this irreversibility is
not known.
By use of different concentrations of very dilute silver ion, t,he
enzymes can be inactivated to varying degrees. It is interesting
to compare the quantities of Ag+ which cause 50 per cent inactiva-
tion of a given amount of the different enzyme preparations.
These quantities, calculated from the data in Tables I and II, are
2.0 X lo-+ milliequivalent of Ag+ per mg. of asclepain m, and
2.5 X 10d6 milliequivalent of Ag+ per mg. of bromelin. From
other published data, the corresponding values for asclepain s
(3) and papain (15) were calculated to be 0.8 X lop5 and 1.1 X 1O-5
milliequivalent of Ag+ per mg. of enzyme, respectively.
Xolanain-The results of activation-inhibition experiments with
solanain are given in Table IV. It is seen that the behavior of
this protease is very different from that of the previous enzymes.
Reducing agents, such as cysteine, cyanide, and H2S, have no
significant effect on the activity of solanain. Relatively concen-
trated solutions of oxidants such as hydrogen peroxide and ferri-
cyanide fail to inactivate the enzyme. Iodine in neutral solution
 D. M. Greenberg and T. Winnick 771

does inactivate solanain, but it appears unlikely that the reaction

is one of oxidation. Iodoacetic acid, maleic acid, and cuprous
oxide, which react with sulfhydryl compounds, and which com-
monly inactivate papainases, have no effect on the activity of
solanain. Phenylhydrazine, which activates papain (16) and
asclepain s (3), is likewise without effect.

TABLE IV
Activation-Inhibition of Solanain
The untreated enzyme solution, diluted with 2 volumes of water, pro-
duced 0.275 to 0.292 milliequivalent of non-protein nitrogen in 6 ml. of

Downloaded from http://www.jbc.org/ by guest on January 21, 2020

digestion mixture.

Reagent added to enzyme solution 1Ratio of activity to

that of untreated
Inhibitor Activator enzyme

None 0.04 M cysteine 1.6 -1.12

‘1 0.1-0.5 M NaCN 1.10-1.06
‘I 0.1 M H,S 1.06
0.033 M Hz02 None 0.96
0.02 “ K,Fe(CN)s “ 0.98
‘I
0.01 “ I* 0.07
Same 0.1 M NaCN 0.13
0.03 M iodoacetic acid None 0.98
“
0.03 “ maleic acid 0.98
5 mg. CUZO I‘ 1.00
0.02 M phenylhydrazine “
1.03
“
0.2 “ NaN02, pH 3.8 0.50
Acetate buffer, pH 3.8 ‘I 0.94
0.01 N Ag+ or Hg++ “ 0.01-0.06
“
0.002 “ “ 0.14
0.001 “ “ or Hg++ ‘I 0.30-0.34
0.0004 N Ag+ ‘I 0.56
0.01 N Ag+ or Hg++ 0.1 M H&l or 0.1 M NaCN 0.000.13

Philpot and Small (17) have shown that nitrous acid acts on
pepsin to produce a yellow diazo compound which has 50 per cent
of the original activity. It is interesting to note that HNOz
(liberated from NaNOz under comparable conditions) likewise acts
on solanain to form a yellow product which has about half the
original activity. While the nature of the reaction is not known
in the case of solanain, it is possible that phenol groups also are
involved as in the case of crystalline pepsin. If this is true, the
772 Plant Proteases. I

irreversible inactivation of solanain by iodine could be due to the

iodination of phenol groups in the enzyme, similar to the reaction
of pepsin (18). Further evidence for this theory is the fact that
ketene (at 0” and pH 5.5) produces 50 per cent inactivation in 5
minutes and complete inactivation of solanain within an hour.
Solanain is inactivated by 0.01 N Ag+ or Hg++. As with the
previous enzymes, the inactivation by these relatively concen-
trated solutions is irreversible. By partial inactivations with more
dilute Ag+ solutions, it is found that 1.6 X 10e4 milliequivalent
of Ag+ causes 50 per cent inactivation of 1 mg. of solanain. This

Downloaded from http://www.jbc.org/ by guest on January 21, 2020

quantity of Ag+ is about 10 times as great as the corresponding
amounts listed for asclepain m, asclepain s, papain, and bromelin.
If it is assumed that the impurities in each enzyme have an approx-
imately equal effect, the higher value for solanain again suggests
that this enzyme has a different active group than the other
proteases.

Technical assistance was furnished by the personnel of the

Works Progress Administration, Official Project 65-l-08-62,
assigned to the University of California.

SUMMARY

1. A study was made of the activation-inhibition reactions of

three partly purified plant proteases, bromelin of pineapp!e,
asclepain m of the milkweed, Asclepias mexicana, and solanain of
the horse-nettle, Solarium elaeagnijolium.
2. The reactions of bromelin and asclepain m resemble those of
papain and asclepain s (protease of Asclepias speciosa), and are
indicative of the presence of sulfhydryl as a group essential to the
activity of these enzymes.
3. Solanain is not affected by oxidizing or reducing agents, or
by reagents which react with -SH groups. This enzyme is,
therefore, not a papainase.
4. The inactivations produced by nitrous acid and ketene indi-
cate that phenolic groups may be essential for the activity of
solanain. The course of the inactivation of papain by ketene is
7 It may be of interest that solanain resembles trypsin and chymo-
trypsin, but differs from all papainases, in that it does not readily turn
casein white during the digestion process.
 D. M. Greenberg and T. Winnick 773
favorable to the view that phenolic as well as -SH groups are
necessary for the activity of this enzyme.
BIBLIOGRAPHY

1. Balls, A. K., and Lineweaver, H., J. Biol. Chem., 130, 669 (1939).
2. Bodansky, A., J. Biol. Chem., 61, 365 (1924).
3. Winnick, T., Davis, A. R., and Greenberg, D. M., J. Gen. Physiol.,
23, 275, 289, 301 (1940).
4. Northrop, J. H., and Kunitz, M., J. Gen. Physiol., 16, 313 (1932).
5. Anson, M. L., J. Gen. Physiol., 22, 79 (1938).
6. Anson, M. L., J. Gen. Physiol., 23, 321 (1940).
7. Hellerman, L., in Cold Spring Harbor symposia on quantitative bi-

Downloaded from http://www.jbc.org/ by guest on January 21, 2020

ology, Cold Spring Harbor, 7, 165 (1939).
8. Balls, A. K., and Lineweaver, H., Nature, 144, 513 (1939).
9. Kellerman, L., Physiol. Rev., 17, 454 (1937).
10. Hellerman, L., and Perkins, M. E., J. BioZ. Chem., 107, 241 (1934).
11. Fruton, J. S., and Bergmann, M., J. BioZ. Chem., 133, 153 (1940).
12. Herriott, R. M., and Northrop, J. H., J. Gen. Physiol., 18, 35 (1934).
Herriott, R. M., J. Gen. Physiol., 19, 283 (1935).
13. Maschmann, E., Biochem. Z., 279, 225 (1935).
14. Morgan, E. J., and Friedmann, E., Biochem. J., 32, 862 (1938).
15. Krebs, H. A., Biochem. Z., 220, 289 (1930).
16. Balls, A. K., and Hoover, S. R., J. BioZ. Chem., 121,737 (1937).
17. Philpot, J. St. L., and Small, P. A., Biochem. J., 32, 542 (1938).
18. Herriott, R. M., J. Gen. Physiol., 20, 335 (1937).
 PLANT PROTEASES: I.
ACTIVATION-INHIBITION REACTIONS
David M. Greenberg and Theodore Winnick
J. Biol. Chem. 1940, 135:761-773.

Access the most updated version of this article at

http://www.jbc.org/content/135/2/761.citation

Downloaded from http://www.jbc.org/ by guest on January 21, 2020

Alerts:
• When this article is cited
• When a correction for this article is posted

Click here to choose from all of JBC's e-mail

alerts

This article cites 0 references, 0 of which can be

accessed free at
http://www.jbc.org/content/135/2/761.citation.full.h
tml#ref-list-1