ACTIVATION OF ENZYMES*

II. PAPAIN ACTIVITY AS INFLUENCED BY OXIDATION-REDUC- TION AND BY THE ACTION OF METAL COMPOUNDS

BY LESLIE HELLERMAN AND MARIE E. PERKINS

(From the Department of Physiological Chemistry, the Johns Hopkins University, School of Medicine, Baltimore)

(Received for publication, August 1, 1934)

Considerable interest has been displayed recently in the activa- tion and inactivation of enzymes such as arginase, urease, and saccharase. Special impetus to work of this character was given by observations that the rate of proteolysis, catalyzed by papain or liver cathepsins, is enhanced when an organic sulfhydryl com- pound such as glutathione or cysteine is added ’ 3 the reaction mix- tures. S.imilar activations by hydrogen cyanide and by hydrogen sulfide had been known for some time.

A few months ago (1) we reported an apparent relationship be- tween the activity of crystalline urease and its oxidation-reduction state. The controlled treatment of solutions of this hydrolytic enzyme with certain oxidants resulted in the suppression of its activity, and this, in turn, was found to be more or less completely restored by the action of appropriate reducing substances. It was shown further that the urease activity was “extinguished” by the treatment of the enzyme with cuprous oxide or certain organic mercurials (e.g., phenylmercuric hydroxide). This effect was like- wise demonstrated to be reversible. The specificity of these ac- tions and the evidence of Sumner and Poland (2) that crystalline urease contains sulfhydryl as an integral part of its molecule were cited in Paper I to support the tentative hypothesis that urease and possibly certain other enzymes may be reversibly inactivated by oxidation of their sulfhydryl groups, or by the formation of metal- lic derivatives (mercaptides). If the oxidation proceed no further

* The paper entitled “Urease activity as influenced by oxidation and reduction” (1) is considered to be Paper I of this series.

241

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242 Activation of Enzymes. II

than to the formation of the dithio configuration, appropriate re- ducing agents should regenerate the sulfhydryl group and the ac- tivity. Likewise, if the action of a metallic derivative be only the formation of a mercaptide, suitable removal of the metal should restore activity.

That many other enzyme activities may be similarly influenced by the processes of oxidation-reduction is implicit in the literature. In the interpretation of a number of these cases we should expect this working hypothesis to be of value. However, as we shall show in a later paper on arginase, the activation of this enzyme is not so simply explained. On the other hand, the plant proteinase papain (3) furnishes a case which the hypothesis fits. Indeed, it was with papain that Bersin and Logemann and Bersin (4, 5) developed the same hypothesis, published shortly after our first paper on urease.

In this paper, it is shown, by the use of methods similar to those previously employed by us, that papain may readily be inactivated by selected oxidants, notably oxygen, iodine, quinone, and ferri- cyanide ion under such conditions that in each case the action may be reversed by hydrogen sulfide, organic sulfhydryl compounds, and other reducing reagents. The observations of reversibility are the more striking since the proteolytic activity, although fre- quently depressed to zero, can usually be restored and actually carried to the level characteristic of papain completely activated by the reducing reagent used. The inactivations by metallic derivatives and the reversals thereof are shown to parallel closely the corresponding phenomena already demonst,rated for urease.

Procedure

For the sake of clarity we present only a synopsis of our obser- vations with a tabulation of those results that have been repeatedly verified and are considered to be substantially reproducible.

Treatment of Enzyme-Papain (Merck), 19 gm., was added to 600 ml. of water. Conductivity water was used throughout this investigation. The mixture was well stirred, filtered, and papain was recovered by the addition of 1200 ml. of 95 per cent ethanol to the filtrate. The solid was collected at the centrifuge, stirred with 200 ml. of 70 per cent ethanol, collected, redissolved in 200 ml. of water, reprecipitated with 400 ml. of 95 per cent ethanol,

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L. Bellerman and 111. E. Perkins

collected, again stirred with 70 per cent ethanol, and finally col- lected on a Buchner funnel and dried in a vacuum desiccator over phosphorus pentoxide. The product (7.6 gm.) retained constant activity for months.

When to 125 mg. of, papain in 5 ml. of water were added, first, 2 ml. of ammonium hydroxide (1 M) and then 0.5 ml. of 5 per cent sodium nitroprusside, there was obtained a slight coloration (esti- mated by comparative tests equivalent to 5 ml. of 0.0001 M cys- teine or less). The sulfhydryl sulfur present, responding to the nitroprusside test, thus appeared to be of the order lo-’ mole or less. A stronger test was obtained after the papain was first treated with 25 mg. of sodium cyanide and allowed to stand 10 minutes. To the nitroprusside test, as applied here, cysteine and glutathione responded but not ergothioneine and thiosalicylate ion. Sulfhydryl groups, if present in the molecules of papain, are probably not detected by this reagent, as applied here.

Estimation of Proteolytic Activity-An exploratory study of vari- ous standard methods led us to select a modified Sorensen titration procedure as most convenient and sufficiently accurate for the requirements of this investigation. The following details are illustrative. To 50 mg. of papain, which had been triturated with 1 ml. of water and transferred to a 25 ml. volumetric flask, there first were added 5 ml. of 0.2 M sodium citrate buffer, pH 5, and then 10 ml. of an aqueous solution containing 1 gm. of gelatin. The solutions were at 37” when mixed and the mixture, diluted with water to 23.0 ml., was incubated at the same t,emperature. After l$ hours, 1 ml. of formaldehyde (37 per cent) was added and the volume brought to exactly 25 ml. Duplicate titrations were then made upon 10 ml. portions, each of which was diluted wjth 25 ml. of water, treated with 1 ml. of 37 per cent formaldehyde, and rapidly titrated with 0.1 N or 0.2 N sodium hydroxide to the end-point with thymolphthalein (10 drops, 0,04 per cent). From this titer was subtracted the sum of the averages of control esti- mations of buffer plus gelatin (without papain) and of papain alone (i.e. papain plus buffer minus the titration value of the buffer), carried out according to the procedure outlined.

When the effect of added reagents (activators, inactivators, etc.) was tested, appropriate ’ corrections, experimentally ascer- tained by means of adequate controls, were always applied for

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244 Activation of Enzymes. II

any contributions of these reagents per se (e.g. HI) to total acidity. Precautions were taken to insure substantial constancy of hydro- gen ion concentration (pH 5) for the many digestions. When acidic reagents were used, appropriate amounts of sodium car- bonate were added. Experiments were frequently repeated with the use of relatively large amounts of 1 M buffer. All digestions were carried out at 37”. Time-activity curves were worked out with varying amounts of the enzyme preparation (25 to 100 mg.) and under a variety of conditions (enzyme non-activated and ac-

TABLE I

Relative Activities of Papain Preparations

No activator added Activator added NaOH, 0.2 N, re-

Papain wired after digea- tion time

NaOH, 0.2 N,. re- p*lWZ%r23- quired after dlges-

tion Reagent’ tion time

12 hrs. 2 hrs. ,$, lf hrs. 2 hrs. h;;t --~~ -~~

mg. ml. ml. ml. ml. ml. ml. A. 100 1.54 HzS-water,t 5 ml. 2.55 B. 50 0.85 1.02 2.08 Same 1.92$ 2.15 3.39 “ 50 1.02 HCN, 0.02 (pH M 5), 4 ml. 2.19 c. 50 1.03 HzS-water, 5 ml. 2.04

* Added to papain-buffer-gelatin prior to incubation. t HzS-water, saturated; approximately 0.1 M. 1 This value was unchanged when the H2S was permitted to act 10

minutes and then was removed in a stream of Nz prior to the addition of gelatin and incubation. The small contributions of H*S to “total acidity” were ascertained by means of controls.

tivated). In this way it was established that the comparisons of papain activities on the basis of the action upon gelat.in at 37” for 1: hours or more were fully significant and adequate for the purposes of this investigation. Our preliminary supplementary experiments in other directions suggest that a more elaborate study involving variations in substrates, buffer systems, tempera- tures, time of action, etc., would disclose many interesting facts regarding the activation behavior of papain without, however, significantly altering the main condlusions of the present study.

The titration method used was found convenient and sufficiently

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L. Hellerman and M. E. Perkins 245

accurate for comparative purposes. The amount of formalde- hyde used is small compared with the concentrations recommended by Richardson (6) and others. The magnitudes of the titration values recorded are, doubtless, of themselves, of limited value for the estimation of the absolute degree of proteolysis. However, occasional control titrations with more formaldehyde (5 to 9 per cent of final volume), with thymolphthalein or phenolphthalein as indicator, gave results of the same order of magnitude as those recorded.

There are recorded in Table I pertinent data on control estima- tions of the activity of various papain preparations for comparison with the results of inactivations and reactivations given in Tables II to v.

Discussion of Tabulated Results

Action of Cuprous Oxide and Mercuri-Organic Derivatives- Typical results from this phase of the investigation are summarized in Table II. Cuprous oxide is known to be exceptionally useful for the isolation or characterization, as cuprous mercaptides, of sulfhydryl compounds of biological origin, notably glutathione (7) and ergothioneine (8). Its action upon papain is rapid and complete. Equally striking is the inactivating effect of mercuri- organic derivatives of the type RHgX (X = Cl, OH, etc.). It has been proved by several investigators (cf. (9)) that such compounds combine with mercaptans to form substituted mercaptides of the type, RHg-SR’. That the full enzymatic activity is found to reside in latent form in the @rates from excess cuprous oxide and from the mercury derivatives of type RHgX appears to exclude in these experiments the probability of inactivation by adsorption and argues, on the other hand, for the assumption of some kind of chemical attack upon the enzyme system. Pertinent, in this con- nection, is the finding that the disubstituted derivatives bjs- iodomethylmercury (10) or bis-p-tolylmercury introduced no sub- stantial inactivation. Evidently, also, the reactive organically bound halogen atoms of the bis-iodo compound had no marked effect. Hydrogen sulfide and hydrogen cyanide, unlike titanous ions (or complex ions derived from them), were found effective in reversing fully these inactivations and bringing the activity to its maximum.

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246 Activation of Enzymes. II

TABLE II

Reversible Inactivation of Papain by Red Cuprous Oxide and by a Variety of Mercuri-Organic Derivatives

Reagent added

Cuprous oxide, CuzOt$. .............. Same + HzS§. ........................ Phenylmercuric hydroxide,

CsHSHgOHII$. ...................... Same + H2S. ......................... Phenylmercuric chloride, C6H5HgCI,

6.3 mg.71.. ........................ Same .................................

“ + H,S .......................... “ + “ .......................... “ f HCN, 0.02 M, 4 ml. .......... “ + Tic&, 0.02 “ 2 “ ..........

Chloromethylmercuric chloride (lo), C1CH2HgCl, 5.7 mg. 71.. ............

Same + HzS ......................... “ yp*. ............................ “ + HzS ..........................

Bisiodomethylmercury (IO), ICH2HgCH,I, 4.8 mg. T[**. ..........

Same + H2S .......................... Bis-p-tolylmercury, (CLH&H&Hg,

8mg.T** ...........................

- mg.

A. 100 100

100 100

B. 50 50 50 50 50 50

50 50 50 50

50 50

50

I iges- tion time

\iaOH 0.2 N, oorre- spend- ing to ~roteol-

ysis

bs. ml.

1: 0.00 1: 2.62

l$ 0.00 1t 2.53

1: 18;

l$ IS+

2 2

0.00 2.17 0.00 2.01

0.86 1.96

0.96

::tp:l initial totiv- ity’

LPPSP nt per ent of H&” aotiv- ity’

100

100

100 100

90

110

100

100

* These “apparent percentages” are given as a convenient means of comparison of the results with those of Table I. They are usually rounded off to the nearest 10 per cent.

t Gelatin, buffer, and papain were mixed, shaken at once (1 minute) with 100 mg. of red oxide for each 100 mg. of papain, and filtered from excess oxide.

$ The clear filtrate (refiltered where necessary) was tested. $ Saturated HzS-water, 5 ml., was added in each case indicated as the

last step prior to incubation. /I Papain plus water (2 ml. for each 100 mg.) was shaken with mercurial,

33 mg. for each 100 mg. of papain, for 1 minute and filtered; buffer and gel- atin were then added as usual.

f Shaken for 1 minute with papain, 50 mg., and water, 1 ml., several portions being made up in one operation if needed; filtered only where indicated (1); buffer and gelatin then added.

** The mixture was not filtered from excess mercurial.

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L. Hellerman and M. E. Perkins 247

Reversible Inactivation of Papain by Oxidants--Papain is not rapidly inactivated by the uncatalyzed action of the oxygen of the air. In the presence of cupric ions or, to a lesser degree, fer-

TABLE III

Inactivation by Iodine and Reactivation by Reducing Agents

NOrmrtl- ity of

iodine* used

0.0023

0.005

0.05

0.1

-

_ -

-

Iodine Reducing agent

ml.

w 3t 3t

3t

3t 3t 3t 3t 38 3§ 311 311 0.2t 0.2t 0.2t 0.2t 0.2f

H&Gwater, 5 ml. HS-glutathione (neu-

tralized), 30 mg. Thioglycolic acid (neu

tralized), 15 mg.

&S

“

“

<‘

TiCl+ 0.02 M, 2 ml. HCN, 0.02 “ 4 “

&S

Reagents added

-

Papain Diges- p*~p~~~- tion

tion time

mg.

A, 100 100 100

hrs.

100

c. 50 50 50 50 50 50 50 50

B. 50 50 50 50 50

l$ 1t

1t

lf

14 1t la 1: 1t 1; l$ 1: 2 2 2

18) 1st

- T

NaOH 0.2 i-4

ml.

0.45 2.57 2.81

2.75

0.10 2.00 0.03 2.03 0.00 1.00 0.00 1.59 0.00 1.30 1.18 0.00 3.50

.ppar-

zlt”Z nitial :tivit)

30

10

0

0

0

0 125 115

0

ippar- nt per :ent of ‘H*S” ctivity

100 110

110

100

100

50

80

55

100

* Iodine solutions were made up with 5 equivalents of KI. t Iodine added to the mixture buffer-gelatin-papain; the mixture then

stood 15 to 30 minutes at 25” before any other addition. $ The reaction mixture (23 ml.) would have been 0.0003 N with respect

to iodine if the latter had not reacted with the enzyme and gelatin present. $ Iodine added to gelatin-papain mixture which had been brought to

pH 8.5 (with NaOH); the reaction mixture stood 1 hour at 25” before addi- tion of buffer and any other addition.

11 Same as (5) except that gelatin-papain was not alkalinized before addition of iodine.

rous ions, acting presumably as catalysts for oxygenation, inac- tivation may become appreciable (compare Table IV), but is re- versed by the action of hydrogen sulfide, reduced glutathione, and

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248 Activation of Enzymes. II

other appropriate reducing agents. Of interest, in this connec- tion, are Bersin and Logemann’s observations (4) of the reactiva- tion, by glutathione, sulfite ion, or succinodehydrase-succinate, of papain-after inactivation by hydrogen peroxide.

TABLE IV

Aeration Experiments; Action of p-Benzoquinone

Digestion time, 1.25 hours.

Aeration (no addition of metal ions)*. ......... Same + HzS .................................. Aeration (Cu++ added)t ...................... Same + H&S .................................

“ + HS-glutathione (neutralized), 30 mg . “ + thioglycolic acid “ 15 “ . .

Aeration (Fe++ added)$. ..................... Same + H2S ................................. Papain, treated directly with quinoneg ....... Same + H$ ................................. Papain, treated at pH 8.5 with quinonejj ....... Same + H2S. ................................

A “ “ “ Ii LL ‘I (I

C “ ,‘ ‘I

* Papain, 100 mg., plus buffer, vigorously aerated i hour at 25” before addition of gelatin; caprylic alcohol used as antifoam.

t Same as the preceding experiment, except that there were also added, before the aeration, 3 ml. of 0.02 per cent CuSOd.

$ Same as the preceding experiment, except that Feels, 5 mg., was added instead of CuSOa.

Appar- N&H entper 0.2 i-4 3 cent of

initial activitj

ml.

1.75 110 2.58 0.57 40 2.41 2.52 2.69 1.07 70 2.55 0.03 0 1.71 0.09 10 1.74

LPPBP nt per :ent of ‘HZS” :tivity

100

90 100 110

100

80

80

0 Papain, 50 mg. (in 1 ml. of water) plus quinone, 1.3 mg., permitted to stand 1 hour before addition of buffer and gelatin.

/I Same as the preceding experiment, except that the gelatin was added to papain and the mixture brought to pH 8.5 before treatment with quinone.

Somewhat more rapid than oxygen as a depressor of activity is ferricyanide ion. For illustration typical experiments may be cited. Acting upon a solution of 50 mg. of papain, alone, for 1 hour prior to incubation, 0.00001 mole of potassium ferricyanide reduced the apparent activity to 30 per cent of the initial value; the inactivation was less marked (65 per cent) when gelatin and buffer (pH 5) were present during the 1st hour of action. In the

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L. Hellerman and M. E. Perkins

former case, the action of hydrogen sulfide restored the apparent activity to 80 per cent of the maximum and in the latter case, to 90 per cent. In parallel experiments the reductant, ferrocyanide ion, was found not to affect papain activity. When 50 mg. of papain, to which had been added (at pH 5) 0.00004 mole of cys- teine, were treated with 0.00006 mole of potassium ferricyanide, its activity was found to be 100 per cent, and in the absence of the cysteine 65 per cent of the initial value. From this protective action of cysteine, it may be inferred that ferricyanide action upon

TABLE V Latent Activity of Reversibly Inactivated Papain after Initial Period of

Incubation in Buffer-Gelatin Reaction Mixtures

Inactivators introduced by standard methods (Tables II to IV). Activity after initial incubation period (controls), zero. Addition after initial incubation period, 5 ml. of saturated H&S-water. Digestion time after HzS addition, 1.25 hours. Temperature, initial incubation and digestion, 37”.

Inactivator

Cuprous oxide ................. Phenylmercuric hydroxide. ..... Iodine (3 ml., 0.003 N). ......... Quinone (2.5 mg.). ............. Iodine (3 ml., 0.005 N). ......... Phenylmercuric chloride.

.

I I Papain Initial incu preparation bation

period

mg. hrs.

A. 100 1; 100 1; 100 la 100 1;

c. 50 184 50 183

______ ml.

2.43 95 2.45 95 2.41 95 2.14 85 1.73 90 1.86 95

cysteine is decidedly more rapid than upon papain. Other factors cannot be fully evaluated at present.

The remarkably consistent results obtained with iodine and quinone are clearly depicted by the typical data summarized in Tables III and IV. As regards the action of quinone, it may be said that under all of the conditions used, there resulted concomi- tantly with the extensive reversible effect a significant measure of irreversible inactivation. This fact is of added interest when con- sidered in conjunction with the astonishing stability of papain to- ward iodine. However, iodine also induced appreciable irrevers- ible inactivation under well defined conditions. The ability of

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250 Activation of Enzymes. II

added titanous chloride to effect at least significant reactivation (after iodine) seems noteworthy. Of perhaps greater theoretical interest is the failure of hydrocyanic acid to bring the activity to its maximum after the action of iodine (cf. (1)).

Stability of Reversibly Inactivated Papain-That papain, treated with the inactivators used in this work, maintains rather remark- able stability in the presence of the substrate, gelatin, is shown by the data of Table V. After the appropriate application of cu- prous oxide, mercurials, iodine, or quinone the inactivated pro- teinase may be incubated at 37” with buffer and gelatin for 1 to 183 hours, after which the addition of hydrogen sulfide brings about almost maximum reactivation, as shown by the subsequent determination of proteolytic activity.

Action of Various Ions

None of the following salts, under the conditions used, caused an inactivation greater than 15 per cent: manganous sulfate, co- baltous nitrate, nickelous nitrate, ferrous sulfate, magnesium chlo- ride, zinc acetate, palladous chloride, lead acetate, sodium fluoride. In each determination, 0.00003 mole of the salt for 50 mg. of pa- pain was added to papain-buffer-gelatin at pH 5 prior to incuba- tion. The enzyme may have been protected against the action of some of the salts by the buffer present. With mercuric chloride there resulted, as anticipated, inactivation to zero-fully reversible by the action of hydrogen sulfide. The ions, Mn++, Co++, and Niff, in common with Fe++, may be catalysts for the oxygenation of papain. Added with ascorbic acid, ferrous ion apparently ac- tivated papain appreciably, in agreement with the interesting ob- servations of Maschmann and Helmert (11). This effect, in con- tradistinction to that of Fe ++ alone, would be comprehensible in the light of views expressed in this paper if the combination of as- corbic acid with the ions of the reversible Fe++:Fe+++ system con- verted the latter to complex ions of a markedly negative oxidation- reduction system. This possibility is under investigation in this laboratory.

Activation of Papain by Cysteine, Hydrogen Cyanide, and Other Reducing Substtinces. Non-Activation by Ergothioneine

A series of tests of activation (Preparation B, 50 mg.; 2 hours) with cysteine hydrochloride, 0.2 to 12.5 mg., neutralized and added

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L. Hellerman and M. E. Perkins 251

to papain-buffer-gelatin before incubation disclosed the following: with increasing amounts of the activator up to 3.0 mg. (0.006019 .mole) the degree of activation rose rapidly from 40 to 95 per cent of the maximum with cysteine; with 6.3 mg. (0.00004 mole) the activity was maximal (corresponding to 2.90 ml. of 0.2 N NaOH), and was not increased by the addition of more cysteine (to 12.5 mg.). Cysteine activation is thus strongly suggestive of a defi- nite chemical attack (reduction?) upon the system activated, a given quantity of cysteine being apparently equivalent to a defi- nite amount of the enzyme.

Similar series of tests with hydrogen cyanide, 0.02 M, (NaCN solution brought to pH 5 by the addition of HCl) and Tic&, 0.02 M, yielded roughly corresponding results. With 4 ml. of HCN, the activity was approximately maximal (2.19 ml. of 0.2 N NaOH), 2 ml. effecting 90 per cent and 1.0 ml. 80 per cent of the observed maximum activity with this reagent. The maximum activity with hydrogen sulfide under similar conditions corresponded to 2.15 ml. of 0.2 N NaOH. With 2.0 ml. of TiCI3 the activity was 170 per cent of the initial value (1.77 ml. of 0.2 N NaOH). Oxygen was not rigorously excluded in the preliminary tests with TiCI, and the maximal effect of this reagent not determined.

It is of interest that thiosalicylic acid (carefully crystallized) or its ion, CsH4(SH)COO-, in which the sulfhydryl group is carried by a benzene nucleus, is a good activator.

Ergothioneine, unlike cysteine and glutathione, was found in- capable of activating papain. Indeed, its action was somewhat depressant. For this and related studies ergothioneine hydro- chloride was prepared from pig blood by the method of William- son and Meldrum (8) and the free base crystallized according to Benedict, Newton, and Behre (12). Its purity was established by analysis (C, H, N). The inertness of the sulfhydryl sulfur of er- gothioneine (13) in this instance is, perhaps, in keeping with the general behavior of thiolglyoxalines.

GENERAL DISCUSSION

Papain seemingly does not come into equilibrium rapidly with certain strictly reversible systems (e.g. Fe(CN$:Fe(CN)?), the oxidants of which are capable of effecting its reversible inactiva- tion. This is in harmony with the behavior of crystalline urease. Indeed, our unpublished work indicates, in agreement with Sum-

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252 Activation of Enzymes. II

ner’s experience (private communication) that the inactivation of urease by ferricyanide is negligibly slow. Although phenol-indo- phenol and 2,6-dichlorophenol-indophenol were likewise found to depress urease activity inappreciably, p-benzoquinone, the oxidant of a system of comparable potential, under the conditions, de- pressed the urease activity rapidly and irreversibly to zero. This specific irreversible action of quinone in the case of urease and to a lesser degree, papain, may be quite plausibly attributed to an attack through the reactive olefinic groups of the quinone (e.g. addition of -NH, or -SH groups) rather than by a direct oxida- tion. The inactivating effect of quinone upon papain is exten- sively reversible and the reversible portion of the action most prob- ably coincides with oxidation.

In connection with the reversible inactivations of papain by oxygen (with appropriate catalysts) and by iodine, it may be said that the observations are in excellent general agreement with those made upon crystalline urease, although the latter enzyme is more sensitive to irreversible attack by iodine than papain.

The responses of papain activity to the action of the various oxidants used are again suggestive of those to be anticipated were the active enzyme a reduced molecule, En-SH, capable of being reversibly inactivated by controlled oxidation to an inactive dithio form, En-S-S--En. This is depicted as follows:

(oxidation) 2En-SH Ie En-S-S-En + 2(H) (1)

(reduction) (Active) (Inactive)

Sulfhydryl-dithio systems, although reversible oxidation-reduction systems in a broad sense, have not yet been shown experimentally to come into equilibrium rapidly with strictly reversible systems, such as the dye and quinone systems. Their oxidation-reduction chemistry remains, thus far, a specific one. Extending the an- alogy with sulfhydryl chemistry a little further, we might antici- pate, for the reduction of hypothetical dithiopapain by cysteine or glutathione (R-SH) an over-all transformation schematically represented thus :

En-S-S-En + PR-SH t) 2En-SH + R-S-S-R (2)

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L. Hellerman and M. E. Perkins 253

The reducing action of other reagents might introduce slight varia- tions. For example, the full latent activity of oxidized papain would not necessarily be elicited in the first stage of action by hy- drogen cyanide or hydrogen sulfide. The following possibilities may be considered (cf. (14)).

En-S-S-En + HCN --+ En-SH + En-SCN (3)

En-S-S-En + HSH __f En-SH + En-S-SH (4)

Cysteine appears to activate available partially inactivated prep- arations of papain to a much higher level than hydrogen cyanide or hydrogen sulfide (which have comparable effects) and the source of such a divergence may well be sought not only in specific differ- ences in the relative rates of reaction but further in actual varia- tions in the mode of chemical attack. Thus, if Equations 2 to 4 have any validity, it would be anticipated that cysteine should produce more of the active material from a given quantity of oxi- dized papain than hydrogen cyanide or hydrogen sulfide, although secondary actions involving the hydrolysis of such a product as En-SCN or the disproportionation of En-S-SH might slowly yield more of the thiol form. If preparations of papain may be assumed to contain the enzyme system in a partially reduced state, the foregoing considerations very plausibly account for the ob- served differences in the activating abilities of hydrogen cyanide or hydrogen sulfide and of cysteine. Until, however, more direct evidence for the sulfhydryl theory as applied to papain shall have been brought forward, these remarks must be regarded as ten- tative and further discussion is deferred until more exhaustive quantitative work shall have been completed.

The apparent slight content of glutathione or similar compounds in the papain used by us, considered in relation to the higher order of magnitude of cysteine concentration found necessary to induce appreciable activation, makes attractive the assumption that the oxidation and reduction effects reported concern an attack upon the papain molecule itself rather than upon supplementary activa- tors present in the enzyme preparations. Lipmann (15) recently made a similar suggestion in explanation of his findings indicating a reversible oxidative inactivation of glycolytic enzymes by oxygen.

The assumption of a control of activity through the thiol groups

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254 Activation of Enzymes. II

of papain as well as urease seems quite consistent with the obser- vations of reversible effects obtained from the actions of cuprous oxide and organic mercury compounds upon these enzymes. De- tails have already been discussed. For the representation of such actions, we have postulated the following type equations.

2En-SH + CuzO -z 2En-S-Cu + HtO (5)

En-SH + HO-HgGH5 -2 En-S-HgC$Hs + Hz 0 (6)

Assumptions of specific physiological implications of the results of these studies, although tempting, need not be reviewed at this time. Of particular interest is the probability that processes of reversible oxidation-reduction may provide an important mecha- nism for the control in vivo of the activity of intracellular enzymes (hydrolytic and others), influencing, thereby, the metabolism of cells. On the other hand, the observations of the reversible ac- tions of mercuri-organic derivatives may suggest certain ap- proaches to an understanding of the mode of action from a chemi- cal point of view of some chemotherapeutic substances.

SUMMARY

It has been shown that papain may be reversibly inactivated by means of catalyzed but not of uncatalyzed oxygenation and by the action of iodine, quinone, and ferricyanide ion. The reversals are extensive and are effected by a variety of reducing substances. Certain metallic derivatives, notably cuprous oxide and mercurials of the type RHgX, which are known to form with thiol compounds characteristic derivatives (mercaptides), suppress completely the papain activity, which again may be regenerated by the action of appropriate reagents. As a guide to investigation there was em- ployed a working hypothesis which assumed that such effects may be attributed to reversible chemical actions upon the thiol groups of the enzyme molecules themselves. This hypothesis was found useful previously in a similar investigation upon crystalline urease. The activating effects of hydrogen cyanide and particularly cys- teine suggest a stoichiometric relationship in the action of these reagents upon papain. Ergothioneine was found incapable of activating papain under conditions favorable for the action of glutathione and cysteine.

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L. Hellerman and M. E. Perkins

Helpful suggestions generously contributed by Dr. W. M. Clark and Dr. Barnett Cohen are gratefully acknowledged. We also

‘express our appreciation of the assistance of Mr. Robert Broh- Kahn in the preparation of ergothioneine and of the analysis of the latter compound by Dr. R. T. Milner of Washington, D. C.

BIBLIOGRAPHY

1. Hellerman, L., Perkins, M. E., and Clark, W. M., Proc. Nut. Acad. SC., 19, 855 (1933).

2. Sumner, J. B., and Poland, L. O., Proc. Sot. Exp. Biol. and Med., 30, 553 (1933).

3. Review articles on papain and its activation, Grassmann, W., in Oppen- heimer, C., Die Fermente und ihre Wirkungen, Leipsic, 3rd edition, 3, 1012-1021 (1929); Ergebn. Enzymforsch., 1, 152156 (1932).

4. Bersin, T., and Logemann, W., Z. physiol. Chem., 220, 209 (1933). 5. Bersin, T., Z. physiol. Chem., 222, 177 (1933). 6. Richardson, G. M., Proc. Roy. Sot. London, Series B, 116, 121, 142

(1934). 7. Hopkins, F. G., J. Biol. Chem., 84, 269 (1929). Pirie, N. W., Biochem.

J., 26, 614 (1931). 8. Williamson, S. W. W., and Meldrum, N. V., Biochem. J., 26, 815 (1932). 9. Sachs, G., Ann. Chem., 433, 154 (1923).

10. Kellerman, L., and Newman, M. D., J. Am. Chem. SW., 64, 2859 (1932). 11. Maschmann, E., and Helmert, E., Z. physiol. Chem., 223, 127 (1934). 12. Benedict, S. R., Newton, E. B., and Behre, J. A., J. Biol. Chem., 67,

267 (1926). 13. Barger, G., and Ewins, A. J., J. Chem. Sot., 99, 2336 (1911). 14. Mauthner, J., Z. physiol. Chem., 78, 28 (1912). 15. Lipmann, F., Biochem. Z., 266, 133 (1933).

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Leslie Hellerman and Marie E. PerkinsACTION OF METAL COMPOUNDS

OXIDATION-REDUCTION AND BY THEACTIVITY AS INFLUENCED BY

ACTIVATION OF ENZYMES: II. PAPAIN

1934, 107:241-255.J. Biol. Chem. 

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CORRECTIONS

On page 233, Vol. 107, No. 1, October, 1934, Table I, column 3, read Ethyl alcohol, absolute for Methyl alcohol, absolute.

On page 243, Vol. 107, No. 1, October, 1934, line 1, following “ethanol” insert thoroughly washed with 96 per cent ethanol.