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Investigation of snake venom enzymes I. Separation of rattlesnake venom proteinases by cellulose ion-exchange chromatography
482
BIOCHIMICA ET BIOPHYSICA ACTA
INVESTIGATION OF SNAKE VENOM ENZYMES I. S E P A R A T I O N OF R A T T L E S N A K E VENOM P R O T E I N A S E S BY C E L L U L O S E I O N - E X C H A N G E CHROMATOGRAPHY* GERHARD
PFLEIDERER
AND G E O R G E
SUMYK
Institute o/ Organic Chemistry, D3partment o/ Biochemistry, University o/Frank/urt (Germany) (Received F e b r u a r y ist, I96I)
SUMMARY
I. Three different proteolytic enzymes were isolated from the rattlesnake venom by chromatography on DEAE-cellulose. 2. The distinct nature of the proteinases has been confirmed by zone electrophoresis, p H optimum, effect of various ions on activity, substrate specificity, and the mode of action studies. 3. Phosphodiesterase, ATP-pyrophosphatase, 5'-nucleotidase, L-amino acid oxidase, lecithinase, and two fibrinogenclotting activities were also identified in the chromatographic fractions. 4- The presence of the fibrinogen-clotting activity in the venom is discussed.
INTRODUCTION
Although the presence of proteolytic activity in the snake venoms was reported already in the last century by LACERDA 1, a more comprehensive investigation of the enzymes responsible for this activity was only recently initiated. DEUTSCH AND DINIZ2 studied the proteolytic activities of fifteen different snake venoms and concluded that a given venom m a y contain several proteinases. HAMBERG AND ROCHA E SILVA3,4 reported the presence of both heat-stable and heat-labile proteolytic components in the Bothrops jararaca venom, which had benzoylarginine methyl ester and caseinase activities, respectively. HENRIQUES el al. 5,e, have separated two proteolytic fractions from the same venom, one possessing high benzoylarginine amidase activity. HOLTZ AND R A U D O N A T v, found similar situation in the case of B. atrox venom, from which they isolated two components, one strongly proteolytic and the other mainly blood-clotting. For further references see the reviews by SLOTTAs, Z E L L E R 9 and the two recent books 1°, 11 on venoms. The value of proteolytic enzymes as analytical tools in the protein chemistry has induced us to search for new proteinases and to investigate their specificities. During recent years, cellulose ion-exchangers have been used with increasing N o n - s t a n d a r d a b b r e v i a t i o n s : TCA, trichloroacetic acid; L D H , lactic d e h y d r o g e n a s e . * A p r e l i m i n a r y r e p o r t of t h i s w o r k w a s p r e s e n t e d a t t h e m e e t i n g of G e r m a n , F r e n c h , a n d Swiss b i o c h e m i s t s in Zfirich, O c t o b e r io, 196o.
Biochim. Biophys. dcta, 51 (t96I) 482~493
SNAKE VENOM ENZYMES
483
success for the chromatography of proteins. BOMAN .4.N'D KALETTATM achieved a separation of three phosphodiesterases b y chromatography of rattlesnake venom on columns of DEAE-cellulose. Following the fruitless attempts to purify the rattlesnake venom proteinases using fractionation b y adsorption, fractional precipitation and other classical methods, we have successfully employed the DEAE-cellulose chromatography for this purpose. In this communication we report our work on separation and characterization of three proteolytic enzymes from the venom of western diamond-back rattlesnake. MATERIAL AND METHODS
The pooled Crotalus atrox venom was lyophilized following the collection from rattlesnakes, maintained in the pharmacology department serpentarium of the Frankfurt University. I t was stored in the cold and reconstituted immediately before use, by stirring with the starting buffer and centrifugation to remove dissolved material. The venom concentrations used did not exceed 3 % and were not lower than I °/o. Casein, according to HAMMERSTEN was bought from E. Merck A.G., Darmstadt. The I O//ocasein solutions in Tris hydrochloric acid buffer were prepared b y heating for lO-15 min at IOO° until all casein dissolved. Fibrinogen, containing 8 °/o NaC1, was dissolved in 0.05 M Na barbital buffer of p H 7.4 to make 0.2 % solution, 0.85 % in NaC1. Crystalline bovine hemoglobin obtained from Behringwerke, Marbnrg-Lahn, was denatured b y 6 M urea in alkaline solution. The urea was removed by repeated dialysis against the desired buffer. Bovine serum albumin, obtained from the same source as hemoglobin, was oxidized with performic acid at o ° b y the method of HIRS13. Lactic dehydrogenase from pig heart, was prepared according to STRAUB14 and denatured in 4 M urea solution with subsequent dialysis. Crystallized Zinc insulin, 23 IU/mg, was purchased from the Armour Laboratories, Hampden Park, Eastbourne (England). I t was oxidized by the same method as serum albumin. B chain of oxidized insulin was prepared by the method of PIERCE 15, using a 36-tube countercurrent distribution train. The purity was determined electrophoretically and b y chromatographic identification of the ether soluble DNP-amino acids, obtained b y dinitrophenylation and hydrolysis of the peptide according to SANGER'S method 16. Only traces of the DNP-glycine cold be found, indicating that the substrate was relatively free of the A chain or the unoxidized insulin. Trypsin, in very pure crystalline form, was obtained from Novo Industrie G.m.b.H. Pharmaceutika, Mainz, Germany.
Analytical methods The protein content of chromatographic fractions was estimated as the extinction at 280 m/~, using Beckman D K I I recording spectrophotometer. Proteolytic activity was determined b y means of the KUNITZ~7 test. Enzyme aliquots, in small centrifuge tubes, were brought up to i ml with 0.05 M Tris-HC1 buffer of p H 8.2 or 8.9, (depending on the p H - o p t i m u m of the enzyme), and I ml of the I °/o casein solution in the same buffer added. The mixture was incubated for Biocldm. Bicphys. Acta, 51 II96I) 482 493
484
6. PFLEIDERER, G. SUMYK
20 min at 37 ° ; the indigested protein precipitated with 3 ml of 5 % TCA, allowed to stand for 30 min and centrifuged at 3000 rev./min for lO-15 min. The extent of proteolysis was determined by measuring the extinction of the supernatant at 280 m/x against blanks from which the enzyme was omitted. The amount of enzyme solution used in tests was adjusted to give an extinction between 0.05 and 0.3, which lies in the linear portion of the activity ~'s. concentration curve. Fibrinogen-clotting activity of the effluent was followed kinetically. Fibrinogen solution was incubated with the test samples at 37 ° in a thermocuvette and the change in extinction at 405 mr* observed by means of an Eppendorf spectrophotometer. The phosphodiesterase, lecithinase, and L-amino acid oxidase activities were measured as described by BOMAN AND KALETTA 12. 5'-nucleotidase activity was determined as the amount of inorganic phosphate liberated by the enzyme from adenosine monophosphate in a given time at 37 °. A new spectrophotometric method based on a series of biochemical reactions, which will be described in the forthcoming paper, was used for the assay of ATP-pyrophosphatase activity.
The chromatographic technique D E A E - S F cellulose was obtained from Serva Entwicklungslabor, Heidelberg. To improve the flow rate, fine particles of cellulose were removed by sedimenting several times in the starting buffer from 2-3 h and decanting the upper portion. Thus graded cellulose was deaerated, precooled to 2 ° and packed under about 0.2 atm pressure in columns of varying size, depending on the amount of venom to be fractionated. It was found that approx, io g of dry exchanger with a capacity of 0.5-0.6 mequiv./g is sufficient for the separation of IOO mg of crude venom. Before applying the venom solution, the columns were usually washed ovelnight with the starting buffer. They were regenerated by washing with I °/o NaOH followed by the buffer until the effluent reached the desired pH. The chromatograms have been developed by a stepwise or simple gradient elution technique, using Soerensen phosphate buffer of pH 7.2. Linear gradient from o.oi M to 0.20 M concentration was achieved by adding 0.33 M buffer to the mixing chamber containing 700 ml of o.oi M buffer. To calculate the buffer concentration in the eluent the following equation was used: C
=
C O --
Go/eK
C, concentration of buffer, leaving the mixing chamber; Co, Concentration of buffer in the reservoir; K, the ratio of the collected volume of eluent to the volume in mixing chamber. The fractions were collected automatically on the constant volume basis. For small columns (20 × I.O cm) the fraction volume was 3 ml. The entire chromatographic procedure was carried out in the room maintained at 2 ° .
Zone electrophoresis The zone electrophoresis of chromatographic fractions, containing proteolytic activity, was done in starch-gel slurry at about 15 V/cm, using Na barbital buffer of pH 8.6. The protein bands were identified by staining the paper negatives with Amide Black dye.
Biochim. Biophys. Acta, 51 (1961) 482-493
SNAKE VENOM ENZYMES
485
The peptide fragments obtained b y the enzymic digestion of the B chain of insulin were separated by paper electrophoresis at approximately 4 ° V/cm in p H 6.5 pyridine-acetate or p H 1.9 acetate-formate buffers and stained with ninhydrin reagent. p H optima were determined by means of the KUNITZ test in Tris-HC1 buffer system, using casein as the substrate. All enzyme solutions were previously dialysed against water. The substrate concentrations were held constant in every experiment. The activation and inhibition experiments were also based on the caseinase activity determination. The dialysed enzyme solutions were incubated with freshly prepared activators and inhibitors for one hour at room temperature prior to the addition of substrate. The end concentration of the added ions in the incubate was always 2 mM. The substrate specificity determinations, with the exception of the gelatin proteolysis for which the photographic film technique of PHILPOT AND DEUTSCH18 was used, were performed at identical substrate concentrations and conditions employing the KUNITZ test. The enzyme action differences were studied b y investigating the composition of the peptide mixture resulting from substrate hydrolysis by the proteinases. B chain of insulin in 0.05 M NH4HCO 3, adjusted to the optimal p H with ammonia, was digested for lO-15 h at 37 ° b y the freshly prepared proteinases, which have been dialysed against water made 2 m M with respect to CaC12 concentration. The incubates were protected against bacterial attack with few drops of toluene. The lyophilized digests were compared side-by-side electrophoretically. To exclude the possibility of autolysis products contributing to the differences in peptide composition, blanks containing no substrate were run under identical conditions. RESULTS
Chromatography of venom Figs. I and 2 show typical chromatograms made on DEAE-SF cellulose columns. The first was developed b y increasing the buffer concentration stepwise, and the second b y a buffer concentration gradient. The abscissa in the figures is given in tube numbers; solid line represents extinction at 280 m/~; dashed line stands for proteolytic activity. The buffer concentrations are indicated below the abscissa, and other enzymic activities are marked by a line extending throughout the active zone. As can be seen from the figures, three distinct proteolytic zones were obtained. For the sake of convenience they will be referred to as a-, fi-, and ~-proteinases corresponding to the order in which they emerge from the column. In a few cases the middle proteolytic zone was split into two peaks. I t has been found that the first component was eluted b y 0.04 M and the second b y 0.05 M buffers. However, the comparisons of their p H optima, the effect of various ions on their activity, and the peptide fragments from the digests of B insulin chain showed no differences. Therefore, in the future references the two components will be regarded as a single enzyme. The probable cause of this splitting will be discussed later. The total recovery of the crude venom proteolytic activity was about 70 °/'o, with ~,-enzyme as the main portion (70-80 °/o) and the rest of activity approximately equally distributed between the other two proteinases. Biochim. Biophys. Acta, 51 (1961) 482-493
486
G. P F L E I D E R E R , G. SUMYK
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F i g . I. C h r o m a t o g r a m of 200 m g v e n o m of Crotalus atrox o n a 25 × 1.5 c m c o l u m n of D E A E c e l l u l o s e . E l u t i n g b u f f e r w a s p h o s p h a t e a t p H 7.2 w i t h s t e p w i s e c h a n g e s i n c o n c e n t r a t i o n a s i n d i c a t e d b e l o w t h e a b s c i s s a . F r a c t i o n s of 3 m l c o l l e c t e d a t a flow r a t e of a p p r o x i m a t e l y 0.2 m l / m i n . , A b s o r b a n c y a t 28o mff; . . . . . , the proteolytic activity expressed as the extinction at 280 m # g i v e n b y 0.2 m l of t h e e f f l u e n t u n d e r t h e c o n d i t i o n s of t h e I{UNITZ t e s t .
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F i g . 2. C h r o m a t o g r a m of 500 m g v e n o m of Crotalus atrox o n a 30 x 2.2 c m c o l u m n of D E A E c e l l u l o s e . S t a r t i n g b u f f e r o . o I M p h o s p h a t e , p H 7.2. L i n e a r g r a d i e n t t o 0.33 M p h o s p h a t e , p H 7.2. F r a c t i o n s of 6 m l c o l l e c t e d a t a flow r a t e of o. 4 m l / m i n . , a b s o r b a n c y a t 280 m f f ; . . . . . . , t h e p r o t e o l y t i c a c t i v i t y e x p r e s s e d a s t h e e x t i n c t i o n a t 280 m # g i v e n b y 0.2 m l of t h e e f f l u e n t u n d e r t h e c o n d i t i o n s of t h e KIJNITZ t e s t . Biochim. Bi ophy s . Acta, 51 (1961) 4 8 2 - 4 9 3
487
SNAKE VENOM ENZYMES
In view of the manifold biological activity of the snake venoms n, we have tested the effluent for the presence of some other enzymes. Fibrinogen-clotting, phosphodiesterase, 5'-nucleotidase, and ATP-pyrophosphatase activities have been found in the first peak, together with the a-proteinase. The zone eluted by 0.02 M buffer contained a second fibrinogen-clotting enzyme and the L-amino acid oxidase, the latter being easily identified by its fluorescence in ultraviolet light. Lecithinase activity was present in the effluent containing 7-proteinase. Preliminary toxicity tests on mice revealed a toxic component in the first peak. Interestingly, it was observed that the 20-30 tubes of Fig. I gave ultraviolet spectra with tryptophan characteristic shoulders at 285 m/~. The protein concentrations of these fractions estimated by the biuret method was very low in relation to the extinction, indicating presence of tryptophan rich, TCA soluble component(s). When subjected to zone electrophoresis at pH 6.5, this peak showed a band with moderate anode mobility positive to Ehrlich and ninhydrin reagents, and a second
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Fig. 3. E l e c t r o p h e r o g r a m of t h e three proteolytic zones from DEAE-cellulose c h r o m a t o g r a p h y . 2 h in starch-gel s l u r r y at 15 V/cm, p H 8.6. Stained with Amido Black B.
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Fig. 4. Effect of p H on the proteolytic a c t i v i t y of the three c o m p o n e n t s isolated from C. a t r o x venom. D e t e r m i n e d b y t h e KUNITZ method, with casein as the s u b s t r a t e in 0.0 5 M Tris-HC1 buffer.
Biochim. Biophys. Acta, 51 (1961) 482-493
488
G. P F L E I D E R E R ,
G. S U M Y K
weak, neutral band stained with Amido Black dye. No toxicity or above mentioned enzyme activities were associated with this chromatographic zone.
Zone electrophoresis Fig. 3 shows an electropherogram of the three proteolytically active zones obtained by developing the DEAE-cellulose column with o.oi M, 0.067 M buffers and a gradient to limit 0.35 M phosphate buffer concentration. The zone containing the a-proteinase is split into at least three distinct protein bands moving slowly toward cathode ; the middle zone shows four to five weak bands moving with varying anode mobilities; the ~-proteinase fraction migrates relatively fast toward anode in two strong, closely associated protein bands.
pH optima Fig. 4 shows the pH-activity curves of the separated proteolytic components. The a- and y-proteinases have optimal activity at pH 8.8-9.0 in Tris-HC1 buffer with casein as the substrate. The fl-enzyme, on the other hand, has an activity m a x i m u m between pH 8.0 and 8.2 in the same system. TABLE
I
EI~FECT OF CATIONS AND ANIONS ON THE ACTIVITY OF THE THREE PROTEINASES t~ROM C r o t a l u s a t r o x VENOM
S o l u t i o n s of i o n s (final c o n c n . 2 m M ) w e r e i n c u b a t e d for i h a t r o o m t e m p e r a t u r e w i t h t h e p r o t e i n a s e s b e f o r e a d d i t i o n of s u b s t r a t e . Relative activity Ion o~
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TABLE
II
COMPARISON OF THE HYDROLYTIC ACTIVITIES OF THE THREE SEPARATED RATTLESNAKE VENOM PROTEINASES ON VARIOUS SUBSTRATES
T h e a c t i v i t i e s w e r e d e t e r m i n e d b y t h e KUNITZ m e t h o d w i t h i d e n t i c a l s u b s t r a t e c o n c e n t r a t i o n . Hydrolysis ratio casein/substrate Substrate
Hemoglobin* Albumin** Insulin** LDH *
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* D e n a t u r e d b y urea. ** O x i d i z e d w i t h p e r f o r m i c acid. Biochim.
Biophys.
Acta,
51 ( 1 9 6 1 ) 4 8 2 - 4 9 3
489
SNAKE VENOM ENZYMES
Effect of ions on enzyme activity As can be seen from Table I the ~- and fl-proteinases are activated by Ca 2+ or Mg 2+ ions. Sulphide ions decrease the activity of these enzymes by 43 % and that of 7 b y 55 o/ /o. All three enzymes are strongly inhibited by CN- and Hg ~+ ions. Furthermore, all of them are completely inactivated by the metal binding agent EDTA.
Substrate specificity D a t a summerizedinTable I I were obtained from proteolytic digestion experiments using various substrates. The results reflect marked differences in the sites of enzymic attack, and (or) in the rates of hydrolysis. These differences are most pronounced when insulin is used as the substrate. Compared to the extent of casein proteolysis the digestion of oxidized insulin is five and a half times greater for r, two and a half times for y, but slightly smaller for the a-proteinase. In addition to the results in Table I I gelatin is hydrolysed by a and fl but not b y the y-enzyme.
Investigation of enzymic action Figs. 5 and 6 show electropherograms of the peptide mixtures obtained by digestion of the insulin peptide chain with the separated rattlesnake venom proteinases. For comparison the tryptic digest of the same substrate is included in Fig. 5. pH B.5
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Fig. 5. E l e c t r o p h e r o g r a m of t h e peptide m i x t u r e o b t a i n e d b y digestion of the B chain of insulin w i t h the three r a t t l e s n a k e v e n o m proteinases and t r y p s i n . P a p e r used, W h a t m a n No. 3, buffer p y r i d i n e - a c e t a t e of p H 6.5, 3 h at 4 ° V/cm. Stained w i t h m i n h y d r i n .
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Fig. 6. E l e c t r o p h e r o g r a m of the peptide mixt u r e s obtained b y digestion of the B chain of insulin w i t h the three r a t t l e s n a k e v e n o m proteinases. P a p e r used, W ' h a t m a n No. 3, buffer a c e t a t e - f o r m a t e of p H 1.9, I h at 4 ° V/cm. Stained with ninhydrin.
Due to space limitation, we have omitted all blanks from the electropherograms since they did not show any pronounced bands corresponding to those derived from the substrate. Since the digestion proceeded almost to completion in all cases the results m a y be compared readily. B i o c h i m . B i o p h y s . A c t a , 51 (1961) 482-493
49 °
G. PFLEIDERER, G. SUMYK DISCUSSION
Interpretation of the chromatograms We have explored several elution variants in our chromatographic experiments and found that best separation resulted when the columns were developed as shown in Fig. I. The use of concentration gradients does not give good resolution of the middle zone nor of the lecithinase and ~,-proteinase enzyme. Moreover, it causes inconvenient broadening of the protein zones. Stepwise developing with o.oi M and 0.067 M buffers is a rapid method; however, the second step compresses all components into a single complex zone. The sporadic splitting of the middle proteolytic zone into two components is most likely due to the differences among the venom batches. It was observed that venom which was freeze-dried immediately after collection showed no splitting of fl-proteinase peak, whereas that which has been allowed to stand at room temperature for a short period before freezing produced chromatograms with subdivided middle proteolytic zone. The same phenomenon was also observed by other investigators. BJ6RK AND BOI~IAN19, studied the effect of collection method on the composition of the cobra venom in reference to phosphodiesterase activity. They found considerable variations among the venom batches dried at room temperature over CaC12 as compared to the "freeze-dried venom". The above authors report the splitting of a chromatographically homogeneous isolated phosphodiesterase into three peaks upon autoincubation. Many other examples of such enzyme heterogeneity may be cited; among the better known are the isozymes of LDH which were investigated in detail by WlELAND AND PFLEIDERER2°. In view of the above facts and the identical enzymic properties of the two components mentioned earlier, it appears that the splitting of the r-enzyme is the result of proteolytic digestion. This seems to be further supported by our finding that the r-fraction looses about 6o °'o of its proteolytic activity when autoincubated for 30 min at 37 °. The L-amino acid oxidase activity in 0.02 M buffer effluent was found to be relatively free of other venom protein material. This suggests modification of the current purification method for this enzyme. DEAE-cellulose chromatography could be used as the first step instead of the uneconomical selective heat denaturation 21. In this way, a lot of valuable biologically active proteins would be saved for further investigation. The presence of two fibrinogen-clotting activities, either as separate enzymes or isozymes, is of special interest since Crotalus atrox venom was believed to be free of such activity. The discrepancy is most likely due to the different assay methods employed by earlier investigators. DEUTSCH AND D I N I Z 2, found no fibrin clot after incubation of fibrinogen solution for 3° sec with various amounts of the venom. The typical kinetic curves in Fig. 7 show the reason why this is the case. It appears that an induction period of several minutes exists in the process of fibrin clot formation. This may be the result of the effect of proteolytic enzyme(s), the time lag between the fibrinogen cleavage and cross linking of the fibrin molecules, or the presence of an inhibitor interfering with the clot formation. The first hypothesis is supported by the fact that C. atrox venom is known to digest both fibrinogen and fibrin ~. Furthermore, CHOPRA, MUKERJEE AND CHOWHAN22 were able to show that the blood-clotting Biochim. Biophys. Acta, 51 (I961) 482 493
491
SNAKE VENOM ENZYMES
activity of the Naja naja L. venom is inversly proportional to the venom concentration. Another possibility is the activation of the fibrinogen-clotting enzyme precursor b y the venom proteinases. The time lag assumption is based on the current theory of thrombin catalysed fibrinogen-fibrin transformation. According to LORAND23 the first stage of this process is a "limited proteolytic action" by which a peptide is split off from the fibrinogen molecule and the profibrin formed. Subsequently, the profibrin molecules are cross, linked to fibrin. The work of JANSZKY24 indicates that the fibrinogen-fibrin conversion by animal venoms depends in principle on the same process. Therefore, the relatively slow rate of fibrin production by C. atrox venom could probably be related to the limited amount of the hydrolytic enzyme as compared to other snake venom. This could cause the scarcity of the profibrin micelles, thus further delaying the clot formation. More evidence and purer enzyme preparations will be required to establish the existence of a specific fibrin inhibitor in the rattlesnake venom.
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Fig. 7. F i b r i n o g e n - c l o t t i n g a c t i v i t y of t h e c h r o m a t o g r a p h i c f r a c t i o n s from C. a/fox v e n o m s h o w n as t h e c h a n g e in a b s o r p t i o n a t 4o5 mff. R e a c t i o n m i x t u r e c o n t a i n e d o.I m l e n z y m e s ol ut i on, 1.15 m l of 0.05 3 I N a b a r b i t a l buffer of p H 7.4 a n d 1.25 m l of o . 2 % fi bri noge n s o l u t i o n in t h e s a m e buffer. I n c u b a t e d a t 37 ° in a i - c m c u v e t t e .
It has been observed that the m a x i m a of ATP-pyrophosphatase and 5'-nucleotidase activities precede the m a x i m u m of ~-proteinase, while that of fibrinogen-clotting activity appears one or two tubes later. Working at a neutral pH, we were not able to find more than one zone of phosphodiesterase activity.
Enzymic properties Our characterization experiments revealed considerable differences, as well as certain degree of overlapping in the properties of the separated rattlesnake venom proteinases. Thus, ~-proteinase differs from the jg-enzyme in its p H optimum, electrophoretic mobility, and the relative power to digest hemoglobin and insulin. The two are similar in reference to the effect of metalions on their activities and the ability to hydrolyze gelatin. The 7-enzyme is different from both y- and/Lproteinases since it is not activated b y Ca 2+ or Mg z+ ions, does not digest gelatin, and has distinct electrophoretic mobility. I t resembles ~ with its p H optimum and proteolysis of hemoglobin. All three enzymes are equally affected by EDTA, sulphide, cyanide and mercuric Biochim. Biophys. Acta, 51 (1961) 482 493
492
G. PFLEIDERER, G. SUMYK
ions. They differ from each other in their specificities with respect to peptide bond cleavage, as shown b y the variations in the peptide fragments they produce from B insulin chain. Although the separated bands were not tested for proteolytic activity, results from our earlier electrophoretic experiments in connection with the solubility of the venom indicated presence of three proteolytic components having different mobilities. The three enzymes are quite stable in phosphate buffer solutions at 2 °. Little loss of activity was observed with two week old preparations. None of these property differences, except perhaps the last one, is in itself a sufficient proof for the distinctness of the three proteinases. However, as a whole the differences comprise strong evidence in favor of this hypothesis. The conclusive proof will be, of course, the direct comparison of the enzyme specificities using synthetic substrates. This, however, must await further purification of the proteinases. To the best of our knowledge this is the first attempt to isolate and purify proteolytic enzymes from the venom of Crotalus species. Our findings are strongly supported b y the recent work of MICHL AND KISS 15. In their electrophoretic study of five different snake venoms, the authors cited above showed that two proteinases are present in the C. adamanteus, three in B. atrox, and two in Vipera lebetina venom. The use of gelatin as the substrate in proteolytic tests could explain identification of only two proteinases, if a third is indeed present in the venom of C. adamanteus On the other hand, GONCALVES AND DEUTSCH 24, proved that the members of Crotalidae family m a y be differentiated by paper electrophoresis and ultracentrifugal analysis; therefore, one might expect certain minor differences in the composition of the two venoms in question. Many investigators 11 assigned the venom proteinases to the tryptic type in reference to their p H optima and peptide substrate specificities. However, it was observed that these enzymes were not affected b y typical trypsin inhibitors. Fig. 5 shows that the rattlesnake venom proteinases act on the insulin peptide chain in entirely different manner from trypsin. Evidently the venom enzymes hydrolyse peptide linkages other than those involving arginine or lysine. In fact, our preliminary study of the specificity indicates that the carboxyl end of leucine peptide linkage is the site of the fl-proteinase attack. The fact that all three venom proteinases are strongly inhibited by EDTA, cyanide and sulphide ions points to their common metal protein nature. In the case of a- and fl-enzymes this was directly confirmed b y the activation with Ca 2+ and Mg ~+ ions. The 7-proteinase activity was not affected by these ions, which indicates either a requirement for other metal or extraordinary binding capacity for the above ions. The strong inhibition of the rattlesnake venom proteinases b y Hg e+ ions shows the presence of sulfhydryl groups necessary for the activity. Further purification of the separated rattlesnake venom proteinases and the study of their specificities is now in progress. ACKNOWLEDGEMENTS
This investigation was supported b y funds from European Research Office, U.S. We wish to express our thanks to Dr. H. W. RAUDONAT for the generous supply of the venom. Biochim. Biophys. Acta, 51 (1961) 482-493
SNAKE
VENOM
ENZYMES
493
REFERENCES 1 j . B. LACERDA, Lemons sur le Venin des Serpents du Brasil, L o b a e r t s and Co., Rio de Janeiro, 1884, p. 125. 2 H . V. DEUTSCH AND C. R . DINIZ, J. Biol. Chem., 216 (1955) 17. 3 U. I-IAMBERG AND M. ROCHA E SILVA, Ciencia e Cult., 8 (1956) 176. 4 U . HAMBERG AND I'Ve. ROCHA E SILVA, Arch. intern. Pharm~codynamie, i i o (1957) 222. 50.
]3. HENRIQUES, A. A. C. LAVRAS, 1~. FICHMAN, F . R . I~ANDELBAUM AND S. B . }tENRIQUES,
Bioehem. J., 6S (1958) 597-605. B. HENRIQUES, A. A. C. LAVRAS AND M. FICHMAN, Ciencia e cult., 8 (1956) 24o. 7 p. HOLTZ AND H. ~¢V. RAUDONAT, Arch. exptl. Pathol, Pharmakol., Naunyn-Schmiedeberg's, 229 (1956) 113. s K. SLOTTA, Experientia, 9 (1953) 81. 9 E. A. ZELLER, in J. B. SUMNER AND K, IV[YRBACK, The Enzymes, \%1. I, Part. 2, Academic Press Inc., New York, 1951, p. 999. 10 E . B . BUCKLEY AND N . PORGES, Venoms, American Association for the A d v a n c e m e n t of Science, "~Vashington, 1956. 11 E. KAISER AND H. MICHL, Die Biochemie der Tierischen Gi]te, F r a n z Deuticke, Vienna, 1958. 12 H. G. BOMAN AND U. I(ALETTA, Biochim. Biophys. Acta, 24 (1957) 619. 13 C. H. \V. HIRS, J. Biol. Chem., 219 (1956) 611. 14 F. ]3. STRAUB, Biochem, J., 34 (194 o) 483 . 1~ j . G. PIERCE, J. Am. Chem. Soc., 77 (1955) 184. 16 F. ~ANGER AND E. O. P . THOMPSON, Biochem. J., 53 (1953) 353. IT M. KUNITZ, J. Gen. Physiol., 3 ° (1946) 291. i8 V. 13. PHILPOT JR. AND H. V. DEUTSCH, Biochim. Biophys. Acta, 21 (1956) 524 . 19 \V. BJ6RK AND H. G. BOMAN, Biochim. Biophys. Acta, 34 (1959) 512. 20 TH. \VIELAND AND G. PFLEIDERER, Biochem. Z., 329 (1957) 112. 21 D. \VELLNER AND A. MEISTER, J. Biol. Chem., 235 (196o) 2o13. 22 R. N. CHOPRA, S. N. MUKERJEE AND J. S. CHOWHAN, fndian J. Med. Research, 25 (1937) 137, 23 L. LORAND, Physiol. Revs., 34 (1954) 742. 24 B. JANSZK¥, Science, 112 (195o) 173. 25 H. MICHL AND G. KISS, Monatsh. Chem., 90 (1959) 6o4. 36 j . 1v[. GONCALVES AND H. F. DEUTSCH, Arch. Biochem. Biophys., 6o (1956) 402, 60.
Biochim. Biophys. Aeta, 51 (1961) 482-493
BIOCHIMICA ET BIOPHYSICA ACTA
INVESTIGATION OF SNAKE VENOM ENZYMES I. S E P A R A T I O N OF R A T T L E S N A K E VENOM P R O T E I N A S E S BY C E L L U L O S E I O N - E X C H A N G E CHROMATOGRAPHY* GERHARD
PFLEIDERER
AND G E O R G E
SUMYK
Institute o/ Organic Chemistry, D3partment o/ Biochemistry, University o/Frank/urt (Germany) (Received F e b r u a r y ist, I96I)
SUMMARY
I. Three different proteolytic enzymes were isolated from the rattlesnake venom by chromatography on DEAE-cellulose. 2. The distinct nature of the proteinases has been confirmed by zone electrophoresis, p H optimum, effect of various ions on activity, substrate specificity, and the mode of action studies. 3. Phosphodiesterase, ATP-pyrophosphatase, 5'-nucleotidase, L-amino acid oxidase, lecithinase, and two fibrinogenclotting activities were also identified in the chromatographic fractions. 4- The presence of the fibrinogen-clotting activity in the venom is discussed.
INTRODUCTION
Although the presence of proteolytic activity in the snake venoms was reported already in the last century by LACERDA 1, a more comprehensive investigation of the enzymes responsible for this activity was only recently initiated. DEUTSCH AND DINIZ2 studied the proteolytic activities of fifteen different snake venoms and concluded that a given venom m a y contain several proteinases. HAMBERG AND ROCHA E SILVA3,4 reported the presence of both heat-stable and heat-labile proteolytic components in the Bothrops jararaca venom, which had benzoylarginine methyl ester and caseinase activities, respectively. HENRIQUES el al. 5,e, have separated two proteolytic fractions from the same venom, one possessing high benzoylarginine amidase activity. HOLTZ AND R A U D O N A T v, found similar situation in the case of B. atrox venom, from which they isolated two components, one strongly proteolytic and the other mainly blood-clotting. For further references see the reviews by SLOTTAs, Z E L L E R 9 and the two recent books 1°, 11 on venoms. The value of proteolytic enzymes as analytical tools in the protein chemistry has induced us to search for new proteinases and to investigate their specificities. During recent years, cellulose ion-exchangers have been used with increasing N o n - s t a n d a r d a b b r e v i a t i o n s : TCA, trichloroacetic acid; L D H , lactic d e h y d r o g e n a s e . * A p r e l i m i n a r y r e p o r t of t h i s w o r k w a s p r e s e n t e d a t t h e m e e t i n g of G e r m a n , F r e n c h , a n d Swiss b i o c h e m i s t s in Zfirich, O c t o b e r io, 196o.
Biochim. Biophys. dcta, 51 (t96I) 482~493
SNAKE VENOM ENZYMES
483
success for the chromatography of proteins. BOMAN .4.N'D KALETTATM achieved a separation of three phosphodiesterases b y chromatography of rattlesnake venom on columns of DEAE-cellulose. Following the fruitless attempts to purify the rattlesnake venom proteinases using fractionation b y adsorption, fractional precipitation and other classical methods, we have successfully employed the DEAE-cellulose chromatography for this purpose. In this communication we report our work on separation and characterization of three proteolytic enzymes from the venom of western diamond-back rattlesnake. MATERIAL AND METHODS
The pooled Crotalus atrox venom was lyophilized following the collection from rattlesnakes, maintained in the pharmacology department serpentarium of the Frankfurt University. I t was stored in the cold and reconstituted immediately before use, by stirring with the starting buffer and centrifugation to remove dissolved material. The venom concentrations used did not exceed 3 % and were not lower than I °/o. Casein, according to HAMMERSTEN was bought from E. Merck A.G., Darmstadt. The I O//ocasein solutions in Tris hydrochloric acid buffer were prepared b y heating for lO-15 min at IOO° until all casein dissolved. Fibrinogen, containing 8 °/o NaC1, was dissolved in 0.05 M Na barbital buffer of p H 7.4 to make 0.2 % solution, 0.85 % in NaC1. Crystalline bovine hemoglobin obtained from Behringwerke, Marbnrg-Lahn, was denatured b y 6 M urea in alkaline solution. The urea was removed by repeated dialysis against the desired buffer. Bovine serum albumin, obtained from the same source as hemoglobin, was oxidized with performic acid at o ° b y the method of HIRS13. Lactic dehydrogenase from pig heart, was prepared according to STRAUB14 and denatured in 4 M urea solution with subsequent dialysis. Crystallized Zinc insulin, 23 IU/mg, was purchased from the Armour Laboratories, Hampden Park, Eastbourne (England). I t was oxidized by the same method as serum albumin. B chain of oxidized insulin was prepared by the method of PIERCE 15, using a 36-tube countercurrent distribution train. The purity was determined electrophoretically and b y chromatographic identification of the ether soluble DNP-amino acids, obtained b y dinitrophenylation and hydrolysis of the peptide according to SANGER'S method 16. Only traces of the DNP-glycine cold be found, indicating that the substrate was relatively free of the A chain or the unoxidized insulin. Trypsin, in very pure crystalline form, was obtained from Novo Industrie G.m.b.H. Pharmaceutika, Mainz, Germany.
Analytical methods The protein content of chromatographic fractions was estimated as the extinction at 280 m/~, using Beckman D K I I recording spectrophotometer. Proteolytic activity was determined b y means of the KUNITZ~7 test. Enzyme aliquots, in small centrifuge tubes, were brought up to i ml with 0.05 M Tris-HC1 buffer of p H 8.2 or 8.9, (depending on the p H - o p t i m u m of the enzyme), and I ml of the I °/o casein solution in the same buffer added. The mixture was incubated for Biocldm. Bicphys. Acta, 51 II96I) 482 493
484
6. PFLEIDERER, G. SUMYK
20 min at 37 ° ; the indigested protein precipitated with 3 ml of 5 % TCA, allowed to stand for 30 min and centrifuged at 3000 rev./min for lO-15 min. The extent of proteolysis was determined by measuring the extinction of the supernatant at 280 m/x against blanks from which the enzyme was omitted. The amount of enzyme solution used in tests was adjusted to give an extinction between 0.05 and 0.3, which lies in the linear portion of the activity ~'s. concentration curve. Fibrinogen-clotting activity of the effluent was followed kinetically. Fibrinogen solution was incubated with the test samples at 37 ° in a thermocuvette and the change in extinction at 405 mr* observed by means of an Eppendorf spectrophotometer. The phosphodiesterase, lecithinase, and L-amino acid oxidase activities were measured as described by BOMAN AND KALETTA 12. 5'-nucleotidase activity was determined as the amount of inorganic phosphate liberated by the enzyme from adenosine monophosphate in a given time at 37 °. A new spectrophotometric method based on a series of biochemical reactions, which will be described in the forthcoming paper, was used for the assay of ATP-pyrophosphatase activity.
The chromatographic technique D E A E - S F cellulose was obtained from Serva Entwicklungslabor, Heidelberg. To improve the flow rate, fine particles of cellulose were removed by sedimenting several times in the starting buffer from 2-3 h and decanting the upper portion. Thus graded cellulose was deaerated, precooled to 2 ° and packed under about 0.2 atm pressure in columns of varying size, depending on the amount of venom to be fractionated. It was found that approx, io g of dry exchanger with a capacity of 0.5-0.6 mequiv./g is sufficient for the separation of IOO mg of crude venom. Before applying the venom solution, the columns were usually washed ovelnight with the starting buffer. They were regenerated by washing with I °/o NaOH followed by the buffer until the effluent reached the desired pH. The chromatograms have been developed by a stepwise or simple gradient elution technique, using Soerensen phosphate buffer of pH 7.2. Linear gradient from o.oi M to 0.20 M concentration was achieved by adding 0.33 M buffer to the mixing chamber containing 700 ml of o.oi M buffer. To calculate the buffer concentration in the eluent the following equation was used: C
=
C O --
Go/eK
C, concentration of buffer, leaving the mixing chamber; Co, Concentration of buffer in the reservoir; K, the ratio of the collected volume of eluent to the volume in mixing chamber. The fractions were collected automatically on the constant volume basis. For small columns (20 × I.O cm) the fraction volume was 3 ml. The entire chromatographic procedure was carried out in the room maintained at 2 ° .
Zone electrophoresis The zone electrophoresis of chromatographic fractions, containing proteolytic activity, was done in starch-gel slurry at about 15 V/cm, using Na barbital buffer of pH 8.6. The protein bands were identified by staining the paper negatives with Amide Black dye.
Biochim. Biophys. Acta, 51 (1961) 482-493
SNAKE VENOM ENZYMES
485
The peptide fragments obtained b y the enzymic digestion of the B chain of insulin were separated by paper electrophoresis at approximately 4 ° V/cm in p H 6.5 pyridine-acetate or p H 1.9 acetate-formate buffers and stained with ninhydrin reagent. p H optima were determined by means of the KUNITZ test in Tris-HC1 buffer system, using casein as the substrate. All enzyme solutions were previously dialysed against water. The substrate concentrations were held constant in every experiment. The activation and inhibition experiments were also based on the caseinase activity determination. The dialysed enzyme solutions were incubated with freshly prepared activators and inhibitors for one hour at room temperature prior to the addition of substrate. The end concentration of the added ions in the incubate was always 2 mM. The substrate specificity determinations, with the exception of the gelatin proteolysis for which the photographic film technique of PHILPOT AND DEUTSCH18 was used, were performed at identical substrate concentrations and conditions employing the KUNITZ test. The enzyme action differences were studied b y investigating the composition of the peptide mixture resulting from substrate hydrolysis by the proteinases. B chain of insulin in 0.05 M NH4HCO 3, adjusted to the optimal p H with ammonia, was digested for lO-15 h at 37 ° b y the freshly prepared proteinases, which have been dialysed against water made 2 m M with respect to CaC12 concentration. The incubates were protected against bacterial attack with few drops of toluene. The lyophilized digests were compared side-by-side electrophoretically. To exclude the possibility of autolysis products contributing to the differences in peptide composition, blanks containing no substrate were run under identical conditions. RESULTS
Chromatography of venom Figs. I and 2 show typical chromatograms made on DEAE-SF cellulose columns. The first was developed b y increasing the buffer concentration stepwise, and the second b y a buffer concentration gradient. The abscissa in the figures is given in tube numbers; solid line represents extinction at 280 m/~; dashed line stands for proteolytic activity. The buffer concentrations are indicated below the abscissa, and other enzymic activities are marked by a line extending throughout the active zone. As can be seen from the figures, three distinct proteolytic zones were obtained. For the sake of convenience they will be referred to as a-, fi-, and ~-proteinases corresponding to the order in which they emerge from the column. In a few cases the middle proteolytic zone was split into two peaks. I t has been found that the first component was eluted b y 0.04 M and the second b y 0.05 M buffers. However, the comparisons of their p H optima, the effect of various ions on their activity, and the peptide fragments from the digests of B insulin chain showed no differences. Therefore, in the future references the two components will be regarded as a single enzyme. The probable cause of this splitting will be discussed later. The total recovery of the crude venom proteolytic activity was about 70 °/'o, with ~,-enzyme as the main portion (70-80 °/o) and the rest of activity approximately equally distributed between the other two proteinases. Biochim. Biophys. Acta, 51 (1961) 482-493
486
G. P F L E I D E R E R , G. SUMYK
0.7 §%Nucllotidtst Phosphodlesttr ate AT P- pyropholphtddts. Flbrlnogen - C04,gulisl
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F i g . I. C h r o m a t o g r a m of 200 m g v e n o m of Crotalus atrox o n a 25 × 1.5 c m c o l u m n of D E A E c e l l u l o s e . E l u t i n g b u f f e r w a s p h o s p h a t e a t p H 7.2 w i t h s t e p w i s e c h a n g e s i n c o n c e n t r a t i o n a s i n d i c a t e d b e l o w t h e a b s c i s s a . F r a c t i o n s of 3 m l c o l l e c t e d a t a flow r a t e of a p p r o x i m a t e l y 0.2 m l / m i n . , A b s o r b a n c y a t 28o mff; . . . . . , the proteolytic activity expressed as the extinction at 280 m # g i v e n b y 0.2 m l of t h e e f f l u e n t u n d e r t h e c o n d i t i o n s of t h e I{UNITZ t e s t .
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F i g . 2. C h r o m a t o g r a m of 500 m g v e n o m of Crotalus atrox o n a 30 x 2.2 c m c o l u m n of D E A E c e l l u l o s e . S t a r t i n g b u f f e r o . o I M p h o s p h a t e , p H 7.2. L i n e a r g r a d i e n t t o 0.33 M p h o s p h a t e , p H 7.2. F r a c t i o n s of 6 m l c o l l e c t e d a t a flow r a t e of o. 4 m l / m i n . , a b s o r b a n c y a t 280 m f f ; . . . . . . , t h e p r o t e o l y t i c a c t i v i t y e x p r e s s e d a s t h e e x t i n c t i o n a t 280 m # g i v e n b y 0.2 m l of t h e e f f l u e n t u n d e r t h e c o n d i t i o n s of t h e KIJNITZ t e s t . Biochim. Bi ophy s . Acta, 51 (1961) 4 8 2 - 4 9 3
487
SNAKE VENOM ENZYMES
In view of the manifold biological activity of the snake venoms n, we have tested the effluent for the presence of some other enzymes. Fibrinogen-clotting, phosphodiesterase, 5'-nucleotidase, and ATP-pyrophosphatase activities have been found in the first peak, together with the a-proteinase. The zone eluted by 0.02 M buffer contained a second fibrinogen-clotting enzyme and the L-amino acid oxidase, the latter being easily identified by its fluorescence in ultraviolet light. Lecithinase activity was present in the effluent containing 7-proteinase. Preliminary toxicity tests on mice revealed a toxic component in the first peak. Interestingly, it was observed that the 20-30 tubes of Fig. I gave ultraviolet spectra with tryptophan characteristic shoulders at 285 m/~. The protein concentrations of these fractions estimated by the biuret method was very low in relation to the extinction, indicating presence of tryptophan rich, TCA soluble component(s). When subjected to zone electrophoresis at pH 6.5, this peak showed a band with moderate anode mobility positive to Ehrlich and ninhydrin reagents, and a second
II I i I,
It ~
II i I
II
LI
•~
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"}~
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Kathodc
Fig. 3. E l e c t r o p h e r o g r a m of t h e three proteolytic zones from DEAE-cellulose c h r o m a t o g r a p h y . 2 h in starch-gel s l u r r y at 15 V/cm, p H 8.6. Stained with Amido Black B.
~2
/ // ~
O,OB
0
\ 0.02
7.0
7.5
8.0
8,5
9.0
9.5
pH
Fig. 4. Effect of p H on the proteolytic a c t i v i t y of the three c o m p o n e n t s isolated from C. a t r o x venom. D e t e r m i n e d b y t h e KUNITZ method, with casein as the s u b s t r a t e in 0.0 5 M Tris-HC1 buffer.
Biochim. Biophys. Acta, 51 (1961) 482-493
488
G. P F L E I D E R E R ,
G. S U M Y K
weak, neutral band stained with Amido Black dye. No toxicity or above mentioned enzyme activities were associated with this chromatographic zone.
Zone electrophoresis Fig. 3 shows an electropherogram of the three proteolytically active zones obtained by developing the DEAE-cellulose column with o.oi M, 0.067 M buffers and a gradient to limit 0.35 M phosphate buffer concentration. The zone containing the a-proteinase is split into at least three distinct protein bands moving slowly toward cathode ; the middle zone shows four to five weak bands moving with varying anode mobilities; the ~-proteinase fraction migrates relatively fast toward anode in two strong, closely associated protein bands.
pH optima Fig. 4 shows the pH-activity curves of the separated proteolytic components. The a- and y-proteinases have optimal activity at pH 8.8-9.0 in Tris-HC1 buffer with casein as the substrate. The fl-enzyme, on the other hand, has an activity m a x i m u m between pH 8.0 and 8.2 in the same system. TABLE
I
EI~FECT OF CATIONS AND ANIONS ON THE ACTIVITY OF THE THREE PROTEINASES t~ROM C r o t a l u s a t r o x VENOM
S o l u t i o n s of i o n s (final c o n c n . 2 m M ) w e r e i n c u b a t e d for i h a t r o o m t e m p e r a t u r e w i t h t h e p r o t e i n a s e s b e f o r e a d d i t i o n of s u b s t r a t e . Relative activity Ion o~
fl
V
None
I OO
I OO
I OO
C a 2+ M g 2+
I43 I4 °
136 129
93 IOO
$2CNH g ~+
57 o o
57 o 4
45 o o
TABLE
II
COMPARISON OF THE HYDROLYTIC ACTIVITIES OF THE THREE SEPARATED RATTLESNAKE VENOM PROTEINASES ON VARIOUS SUBSTRATES
T h e a c t i v i t i e s w e r e d e t e r m i n e d b y t h e KUNITZ m e t h o d w i t h i d e n t i c a l s u b s t r a t e c o n c e n t r a t i o n . Hydrolysis ratio casein/substrate Substrate
Hemoglobin* Albumin** Insulin** LDH *
*¢
fl
1.73 I. 15 1.3 ° 2. IO
0.66 I. 18 o.18 2.9o
"/
1.35 5"75 o.41 I 1.5
* D e n a t u r e d b y urea. ** O x i d i z e d w i t h p e r f o r m i c acid. Biochim.
Biophys.
Acta,
51 ( 1 9 6 1 ) 4 8 2 - 4 9 3
489
SNAKE VENOM ENZYMES
Effect of ions on enzyme activity As can be seen from Table I the ~- and fl-proteinases are activated by Ca 2+ or Mg 2+ ions. Sulphide ions decrease the activity of these enzymes by 43 % and that of 7 b y 55 o/ /o. All three enzymes are strongly inhibited by CN- and Hg ~+ ions. Furthermore, all of them are completely inactivated by the metal binding agent EDTA.
Substrate specificity D a t a summerizedinTable I I were obtained from proteolytic digestion experiments using various substrates. The results reflect marked differences in the sites of enzymic attack, and (or) in the rates of hydrolysis. These differences are most pronounced when insulin is used as the substrate. Compared to the extent of casein proteolysis the digestion of oxidized insulin is five and a half times greater for r, two and a half times for y, but slightly smaller for the a-proteinase. In addition to the results in Table I I gelatin is hydrolysed by a and fl but not b y the y-enzyme.
Investigation of enzymic action Figs. 5 and 6 show electropherograms of the peptide mixtures obtained by digestion of the insulin peptide chain with the separated rattlesnake venom proteinases. For comparison the tryptic digest of the same substrate is included in Fig. 5. pH B.5
DH 1.9
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)
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i
I
I
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) C"
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) L
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Fig. 5. E l e c t r o p h e r o g r a m of t h e peptide m i x t u r e o b t a i n e d b y digestion of the B chain of insulin w i t h the three r a t t l e s n a k e v e n o m proteinases and t r y p s i n . P a p e r used, W h a t m a n No. 3, buffer p y r i d i n e - a c e t a t e of p H 6.5, 3 h at 4 ° V/cm. Stained w i t h m i n h y d r i n .
ct
/j
~,
Fig. 6. E l e c t r o p h e r o g r a m of the peptide mixt u r e s obtained b y digestion of the B chain of insulin w i t h the three r a t t l e s n a k e v e n o m proteinases. P a p e r used, W ' h a t m a n No. 3, buffer a c e t a t e - f o r m a t e of p H 1.9, I h at 4 ° V/cm. Stained with ninhydrin.
Due to space limitation, we have omitted all blanks from the electropherograms since they did not show any pronounced bands corresponding to those derived from the substrate. Since the digestion proceeded almost to completion in all cases the results m a y be compared readily. B i o c h i m . B i o p h y s . A c t a , 51 (1961) 482-493
49 °
G. PFLEIDERER, G. SUMYK DISCUSSION
Interpretation of the chromatograms We have explored several elution variants in our chromatographic experiments and found that best separation resulted when the columns were developed as shown in Fig. I. The use of concentration gradients does not give good resolution of the middle zone nor of the lecithinase and ~,-proteinase enzyme. Moreover, it causes inconvenient broadening of the protein zones. Stepwise developing with o.oi M and 0.067 M buffers is a rapid method; however, the second step compresses all components into a single complex zone. The sporadic splitting of the middle proteolytic zone into two components is most likely due to the differences among the venom batches. It was observed that venom which was freeze-dried immediately after collection showed no splitting of fl-proteinase peak, whereas that which has been allowed to stand at room temperature for a short period before freezing produced chromatograms with subdivided middle proteolytic zone. The same phenomenon was also observed by other investigators. BJ6RK AND BOI~IAN19, studied the effect of collection method on the composition of the cobra venom in reference to phosphodiesterase activity. They found considerable variations among the venom batches dried at room temperature over CaC12 as compared to the "freeze-dried venom". The above authors report the splitting of a chromatographically homogeneous isolated phosphodiesterase into three peaks upon autoincubation. Many other examples of such enzyme heterogeneity may be cited; among the better known are the isozymes of LDH which were investigated in detail by WlELAND AND PFLEIDERER2°. In view of the above facts and the identical enzymic properties of the two components mentioned earlier, it appears that the splitting of the r-enzyme is the result of proteolytic digestion. This seems to be further supported by our finding that the r-fraction looses about 6o °'o of its proteolytic activity when autoincubated for 30 min at 37 °. The L-amino acid oxidase activity in 0.02 M buffer effluent was found to be relatively free of other venom protein material. This suggests modification of the current purification method for this enzyme. DEAE-cellulose chromatography could be used as the first step instead of the uneconomical selective heat denaturation 21. In this way, a lot of valuable biologically active proteins would be saved for further investigation. The presence of two fibrinogen-clotting activities, either as separate enzymes or isozymes, is of special interest since Crotalus atrox venom was believed to be free of such activity. The discrepancy is most likely due to the different assay methods employed by earlier investigators. DEUTSCH AND D I N I Z 2, found no fibrin clot after incubation of fibrinogen solution for 3° sec with various amounts of the venom. The typical kinetic curves in Fig. 7 show the reason why this is the case. It appears that an induction period of several minutes exists in the process of fibrin clot formation. This may be the result of the effect of proteolytic enzyme(s), the time lag between the fibrinogen cleavage and cross linking of the fibrin molecules, or the presence of an inhibitor interfering with the clot formation. The first hypothesis is supported by the fact that C. atrox venom is known to digest both fibrinogen and fibrin ~. Furthermore, CHOPRA, MUKERJEE AND CHOWHAN22 were able to show that the blood-clotting Biochim. Biophys. Acta, 51 (I961) 482 493
491
SNAKE VENOM ENZYMES
activity of the Naja naja L. venom is inversly proportional to the venom concentration. Another possibility is the activation of the fibrinogen-clotting enzyme precursor b y the venom proteinases. The time lag assumption is based on the current theory of thrombin catalysed fibrinogen-fibrin transformation. According to LORAND23 the first stage of this process is a "limited proteolytic action" by which a peptide is split off from the fibrinogen molecule and the profibrin formed. Subsequently, the profibrin molecules are cross, linked to fibrin. The work of JANSZKY24 indicates that the fibrinogen-fibrin conversion by animal venoms depends in principle on the same process. Therefore, the relatively slow rate of fibrin production by C. atrox venom could probably be related to the limited amount of the hydrolytic enzyme as compared to other snake venom. This could cause the scarcity of the profibrin micelles, thus further delaying the clot formation. More evidence and purer enzyme preparations will be required to establish the existence of a specific fibrin inhibitor in the rattlesnake venom.
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Fig. 7. F i b r i n o g e n - c l o t t i n g a c t i v i t y of t h e c h r o m a t o g r a p h i c f r a c t i o n s from C. a/fox v e n o m s h o w n as t h e c h a n g e in a b s o r p t i o n a t 4o5 mff. R e a c t i o n m i x t u r e c o n t a i n e d o.I m l e n z y m e s ol ut i on, 1.15 m l of 0.05 3 I N a b a r b i t a l buffer of p H 7.4 a n d 1.25 m l of o . 2 % fi bri noge n s o l u t i o n in t h e s a m e buffer. I n c u b a t e d a t 37 ° in a i - c m c u v e t t e .
It has been observed that the m a x i m a of ATP-pyrophosphatase and 5'-nucleotidase activities precede the m a x i m u m of ~-proteinase, while that of fibrinogen-clotting activity appears one or two tubes later. Working at a neutral pH, we were not able to find more than one zone of phosphodiesterase activity.
Enzymic properties Our characterization experiments revealed considerable differences, as well as certain degree of overlapping in the properties of the separated rattlesnake venom proteinases. Thus, ~-proteinase differs from the jg-enzyme in its p H optimum, electrophoretic mobility, and the relative power to digest hemoglobin and insulin. The two are similar in reference to the effect of metalions on their activities and the ability to hydrolyze gelatin. The 7-enzyme is different from both y- and/Lproteinases since it is not activated b y Ca 2+ or Mg z+ ions, does not digest gelatin, and has distinct electrophoretic mobility. I t resembles ~ with its p H optimum and proteolysis of hemoglobin. All three enzymes are equally affected by EDTA, sulphide, cyanide and mercuric Biochim. Biophys. Acta, 51 (1961) 482 493
492
G. PFLEIDERER, G. SUMYK
ions. They differ from each other in their specificities with respect to peptide bond cleavage, as shown b y the variations in the peptide fragments they produce from B insulin chain. Although the separated bands were not tested for proteolytic activity, results from our earlier electrophoretic experiments in connection with the solubility of the venom indicated presence of three proteolytic components having different mobilities. The three enzymes are quite stable in phosphate buffer solutions at 2 °. Little loss of activity was observed with two week old preparations. None of these property differences, except perhaps the last one, is in itself a sufficient proof for the distinctness of the three proteinases. However, as a whole the differences comprise strong evidence in favor of this hypothesis. The conclusive proof will be, of course, the direct comparison of the enzyme specificities using synthetic substrates. This, however, must await further purification of the proteinases. To the best of our knowledge this is the first attempt to isolate and purify proteolytic enzymes from the venom of Crotalus species. Our findings are strongly supported b y the recent work of MICHL AND KISS 15. In their electrophoretic study of five different snake venoms, the authors cited above showed that two proteinases are present in the C. adamanteus, three in B. atrox, and two in Vipera lebetina venom. The use of gelatin as the substrate in proteolytic tests could explain identification of only two proteinases, if a third is indeed present in the venom of C. adamanteus On the other hand, GONCALVES AND DEUTSCH 24, proved that the members of Crotalidae family m a y be differentiated by paper electrophoresis and ultracentrifugal analysis; therefore, one might expect certain minor differences in the composition of the two venoms in question. Many investigators 11 assigned the venom proteinases to the tryptic type in reference to their p H optima and peptide substrate specificities. However, it was observed that these enzymes were not affected b y typical trypsin inhibitors. Fig. 5 shows that the rattlesnake venom proteinases act on the insulin peptide chain in entirely different manner from trypsin. Evidently the venom enzymes hydrolyse peptide linkages other than those involving arginine or lysine. In fact, our preliminary study of the specificity indicates that the carboxyl end of leucine peptide linkage is the site of the fl-proteinase attack. The fact that all three venom proteinases are strongly inhibited by EDTA, cyanide and sulphide ions points to their common metal protein nature. In the case of a- and fl-enzymes this was directly confirmed b y the activation with Ca 2+ and Mg ~+ ions. The 7-proteinase activity was not affected by these ions, which indicates either a requirement for other metal or extraordinary binding capacity for the above ions. The strong inhibition of the rattlesnake venom proteinases b y Hg e+ ions shows the presence of sulfhydryl groups necessary for the activity. Further purification of the separated rattlesnake venom proteinases and the study of their specificities is now in progress. ACKNOWLEDGEMENTS
This investigation was supported b y funds from European Research Office, U.S. We wish to express our thanks to Dr. H. W. RAUDONAT for the generous supply of the venom. Biochim. Biophys. Acta, 51 (1961) 482-493
SNAKE
VENOM
ENZYMES
493
REFERENCES 1 j . B. LACERDA, Lemons sur le Venin des Serpents du Brasil, L o b a e r t s and Co., Rio de Janeiro, 1884, p. 125. 2 H . V. DEUTSCH AND C. R . DINIZ, J. Biol. Chem., 216 (1955) 17. 3 U. I-IAMBERG AND M. ROCHA E SILVA, Ciencia e Cult., 8 (1956) 176. 4 U . HAMBERG AND I'Ve. ROCHA E SILVA, Arch. intern. Pharm~codynamie, i i o (1957) 222. 50.
]3. HENRIQUES, A. A. C. LAVRAS, 1~. FICHMAN, F . R . I~ANDELBAUM AND S. B . }tENRIQUES,
Bioehem. J., 6S (1958) 597-605. B. HENRIQUES, A. A. C. LAVRAS AND M. FICHMAN, Ciencia e cult., 8 (1956) 24o. 7 p. HOLTZ AND H. ~¢V. RAUDONAT, Arch. exptl. Pathol, Pharmakol., Naunyn-Schmiedeberg's, 229 (1956) 113. s K. SLOTTA, Experientia, 9 (1953) 81. 9 E. A. ZELLER, in J. B. SUMNER AND K, IV[YRBACK, The Enzymes, \%1. I, Part. 2, Academic Press Inc., New York, 1951, p. 999. 10 E . B . BUCKLEY AND N . PORGES, Venoms, American Association for the A d v a n c e m e n t of Science, "~Vashington, 1956. 11 E. KAISER AND H. MICHL, Die Biochemie der Tierischen Gi]te, F r a n z Deuticke, Vienna, 1958. 12 H. G. BOMAN AND U. I(ALETTA, Biochim. Biophys. Acta, 24 (1957) 619. 13 C. H. \V. HIRS, J. Biol. Chem., 219 (1956) 611. 14 F. ]3. STRAUB, Biochem, J., 34 (194 o) 483 . 1~ j . G. PIERCE, J. Am. Chem. Soc., 77 (1955) 184. 16 F. ~ANGER AND E. O. P . THOMPSON, Biochem. J., 53 (1953) 353. IT M. KUNITZ, J. Gen. Physiol., 3 ° (1946) 291. i8 V. 13. PHILPOT JR. AND H. V. DEUTSCH, Biochim. Biophys. Acta, 21 (1956) 524 . 19 \V. BJ6RK AND H. G. BOMAN, Biochim. Biophys. Acta, 34 (1959) 512. 20 TH. \VIELAND AND G. PFLEIDERER, Biochem. Z., 329 (1957) 112. 21 D. \VELLNER AND A. MEISTER, J. Biol. Chem., 235 (196o) 2o13. 22 R. N. CHOPRA, S. N. MUKERJEE AND J. S. CHOWHAN, fndian J. Med. Research, 25 (1937) 137, 23 L. LORAND, Physiol. Revs., 34 (1954) 742. 24 B. JANSZK¥, Science, 112 (195o) 173. 25 H. MICHL AND G. KISS, Monatsh. Chem., 90 (1959) 6o4. 36 j . 1v[. GONCALVES AND H. F. DEUTSCH, Arch. Biochem. Biophys., 6o (1956) 402, 60.
Biochim. Biophys. Aeta, 51 (1961) 482-493