I5o SHORT COMMUNICATIONS

sc 63035 The effect of various sulfites and glycerol on the heat inactivation

of 3~-hydroxysteroid dehydrogenase of rat liver

The dehydrogenating and transhydrogenating activities of the 3a-hydroxysteroid dehydrogenase (3a-hydroxysteroid-NAD(P) oxidoreductase, EC 1.1.1.5o) obtained from the soluble fraction of rat liver was reported to be destroyed on heating at 57 ° for IO rain. The addition of various cofactors, substrates and other compounds did not protect the enzyme preparation. In this study we observed that various sulfites and glycerol had a protective influence when the enzyme preparation was heated under different conditions.

The 3a-hydroxysteroid dehydrogenase was prepared as previously reported z. The fraction precipitated between 0.5 and 0. 7 saturation of (NH4),SO 4 was dissolved in o.oi M phosphate buffer (pH 7.5), and dialyzed against several changes of buffer until the dialyzing medium gave a negative test for ammonia with Nessler's reagent.

TABLE I

R E C O V E R Y OF D E H Y D R O G E N A T I N G A N D T R A N S H Y D R O G E N A T I N G A C T I V I T I E S OF

3 ( % - H Y D R O X Y S T E R O I D D E H Y D R O G E N A S E OF R A T L I V E R A F T E R H E A T T R E A T M E N T

I N T H E P R E S E N C E OF V A R I O U S S U L F I T E S A N D S U L F H Y D R Y L C O M P O U N D S

The final concentrat ion of the compounds in the flasks was I mM, except t ha t of heparin which was o. 5 mg/ml. The method is described in the text. The NAD-dehydrogenat ing activity was assayed in a sys tem containing 2oo/*moles of phosphate buffer (pH 7-5), i /2mole NAD and o.i ml of enzyme mixture (o.7-1.o mg protein) in a total volume of 3 ml. The NADP-dehy- drogenating activity was assayed in a sys tem containing 20o umoles of g lycine-NaOH buffer (pH 9.5), 0 .5/ ,mole NADP, and o.I ml of enzyme mixture (o.7-i .o mg protein) in a total volume of 3 ml. The reaction was initiated by the addition of 35 #g of androsterone dissolved in io /A dioxane to the reaction cuvettes. The control cuvette contained all ingredients except the steroid. The t ranshydrogenat ing sys tem contained 0.6 Kornberg unit of glucose-6-phosphate dehy- drogenase, 5/ ,moles disodium glucose 6-phosphate, 5/zmoles MgC1 v I /*mole NAD, o.o2/ ,mole NADP, and o. 3 ml of enzyme mixture (2.1-3.o mg protein), 20o/ ,moles phosphate buffer (pH 7.5), in a total volume of 3 ml. The reaction was initiated by the addition of 7 #g androsterone dis- solved in IO #l dioxane to the reaction cuvette. The control cuvette contained all ingredients except the steroid. The changes in absorbancy were measured with a Cary spect rophotometer equipped with a o. I -absorbancy slide-wire a t tachment , employing a I -cm light pa th at 34 ° m/z, 22-23 °. One uni t of all three activities represents a change in absorbancy of o.ooi per min. The per cent recovery was calculated from the activities of similarly prepared flasks (control-unheated)

maintained at 4 ° .

Recovery (%)

Compounds N A D- N A D P- Tr ans- dehydroge~a2ing dehydrogenating hydrogenating

Control-Heated 3 16 o NaHSO s I 1 37 i i Na,S~O 4 2o 53 18 Na~S-oO5 3 ° 57 19 K~S2Os 31 5 ° ] 9 Na~SO a 8 20 4 Cysteine o 2 o fl-Mercaptoethanol o 9 o Sodium thioglycolate o 4 o GSH 2 12 o GSSG o 5 2 Ascorbic acid o 6 o Heparin 3 15 o

Biooh im. B i o p h y s . Acta , 89 (1964) 15o-152

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The NAD- and NADP-dehydrogenating and transhydrogenating activities of the enzyme preparation used were 32, 73 and 2.3 units per mg protein, respectively. The various sulfites were prepared as o.I M solutions and adjusted to pH 7.5 with o.i M NaOH. Each reaction flask contained i mM EDTA, io mg enzyme protein, o.i M phosphate buffer (pH 7.5), and I mM sulfites and other compounds in a total volume of 4 ml. The sulfites and other compounds were omitted from the control flasks. The flasks were kept in an ice bath until used. They were incubated at 56-57 ° for 5 min in a Dubnoff shaker, chilled immediately in an ice bath for IO min, and centrifuged at 34 ooo x g for Io min. The snpernatant solutions were assayed for dehydrogenating and transhydrogenating activities. Two control flasks were prepared. One flask was heated as above, and the other kept in the ice bath throughout. The assay systems are described under Table I. The protein was measured by the biuret method s with crystalline bovine serum albumin as standard.

Tile results of a representative experiment on the recovery of the dehydrogenating and transhydrogenating activities of 3a-hydroxysteroid dehydrogenase after heating at 56-57 ° for 5 min in the presence of sulfites, sulfhydryl and disulfide compounds are shown in Table I. At I mM concentration all the sulfites tested exerted a protective influence on the enzyme to the heat-inactivating procedure; whereas the sulflaydryl and disulfide compounds tested had no effect. On the contrary cysteine and fl- mercaptoethanol appeared to increase the lability of the er~zyme to heat denaturation. Glycerol also had a protective effect as shown in Table II . The activities of the enzyme prepation were protected to a greater extent when glycerol and metabisulfite were used together (Tables I I and I I I ) .

We have previously reported that fl-mercaptoethanol inhibited the dehydro- genating and transhydrogenating activities of 3a-hydroxysteroid dehydrogenase s. In the present s tudy sulfites were found to protect the enzyme against heat denatur- ation. The contrasting effects of these two reagents are interesting since they both

T A B L E I I

RECOVERY OF DEHYDROGENATING AND TRANSHYDROGENATING ACTIVITIES OF 3~-HYDROXYSTEROID DEHYDROGENASE OF RAT LIVER AFTER H~AT TREATMENT

IN VARIOUS CONCENTRATIONS OF GLYC]~ROL AND SODIUM METABISULFITE

Glycerol and IWa2S20~ were omitted from the control and control-heated flasks. The procedure is described in tile text and in Table I. The per cent recovery was calculated from the activities

of the control flask kept at 4 ° .

Recovery (%)

Mixtums NA D- NA DP- Tmns- dehydrogenating dehydrog~onaIing kydrogenading

C o n t r o l - H e a t e d 3 8 o 2 0 % g lyce ro l 7 16 8 3 0 % g lyce ro l 19 28 IO 4 o % g l y c e r o l 3 ° 4 ° 20 5 0 % g l y c e r o l 27 39 21 I m M Na,~S20 ~ 17 35 i I I m M N a ~ S 2 O s + 2 o ~/o g l y c e r o l 27 49 22 I m M Na~S~O 5 + 3 ° ~/o g l y c e r o l 47 64 33 I m M Na~S20 ~ + 4 ° % g l y c e r o l 59 77 44 I m M Na~,S20 s + 5 ° ~o g l y c e r o l 66 82 45

B i o c h i m . B i o p h y s . Ac ta , 89 (1964) 15o-152

I 5 2 SHORT COMMUNICATIONS

T A B L E I I I

RECOVERY OF DEHYDROGENATING AND TRANSHYDROGENATING ACTIVITIES OF 3a-HYDROXYSTEROID DEHYDROGENASE AT VARIOUS TIME INTERVALS

DURING HEAT TREATMENT IN THE PRESENCE 01~ GLYCEROL AND METABISULFITE

A series of f lasks were p repared and assayed as descr ibed in the t e x t and Table I. The flasks were i ncuba t ed a t 5 °o for the des igna ted t ime. F l a sk A (control) con ta ined no Na,S ,O 5 or glycerol ; F l a sk B con ta ined I mM Na~S,Os; F l a sk C con ta ined 30% glycerol ; and F l a sk D con ta ined

i mM Na2S,O 5 and 30% glycerol.

Recovery (%)

Time (m~) N . 4 D - ~ k y ~ i n g NADP-~hy~ogenating Tramhydrogenating

A B C D A B C D A B C D

5 83 94 91 95 77 9o 94 98 84 IOO 86 IOO IO 5 ° 65 73 84 6o 78 84 88 5 ° 85 71 86 15 22 47 59 68 45 67 66 78 23 5 ° 57 71 3 ° 6 12 32 45 15 31 51 74 o 4 3 ° 57

react with disulfide bonds. I t is known that fl-mercaptoethanol reduces various disulfide bonds 4. Sulfite, however, reacts primarily with inter-chain disulfide bonds and with occasional intra-chain bonds 5. The reaction with sulfite results in the for- mation of a thiol and sulfonic acid group. The resulting sulfonic acid groups might hinder inter-chain aggregations or reactions on heating and thus retard denaturation. The mechanism responsible for the protective influence of glycerol on protein to heat denaturation is not known. Its influence appears to differ from that of sulfites since their effects were additive when used in combination.

This investigation was supported by a U.S. Public Health Service Research Career Program Award No. I-K3-AM-5517-oI and Grant C-38o 9 from the National Cancer Institute, U.S. Public Health Service.

Division of Clinical Investigation, Sloan-Kettering Institute,

New York, N.Y. (U.S.A.)

SAMUEL S. K O I D E

MARIA T. TORRES

I S. S. KOIDE, Arch. Biochem. Biophys., 1oi (1963) 278. 2 p. H. VON H IPPEL AND D. F. WAUGH, J. Am, Chem. Soc., 77 (1955) 4311. 8 S. S. KOIDE, Steroids, 3 (1964) 85. 4 R . A . PETERS AND :R, W. WAKELIN, Biochem. J., 43 (1948) 45. 5 R. CECIL AND R. G. WAKE, Biochem. J., 82 (1962) 4Ol.

Received February 24th, 1964 Biochim. Biophys. Acta, 89 (1964) 15o-152

sc 63024 N-Benzyloxycarbonyl amino acids as virtual substrates for papain

Various classes of compounds have been shown to serve as substrates for papain (EC 3.44.1o). In addition to proteins, these include synthetic peptides, amides and esters I as well as poly-a-amino acids2, a. In tiffs communication we wish to present

Biochim. Biophys. Acta, 89 (I964) I 5 2 - I 5 5