Specificity of hemorrhagic proteinase from Crotalus atrox (western diamondback rattlesnake) venom

Biochimica et Biophysica Acta 829 (1985) 127-130 Elsevier

127

BBA Report

BBA 30094

Specificity of hemorrhagic proteinase from Crotalus a t r o x (western diamondback rattlesnake) venom Yumiko Komori, Sumio Hagihara and Anthony T. Tu * Department of Biochemistry, Colorado State University, Fort Collins, CO 80523 (U.S.A.) (Received February 6th, 1985)

Key words: Proteolytic enzyme specificity; Snake venom; Fibrinogenase; Proteinase; (C. atrox)

Hemorrhagic proteinase, HTb, isolated from Crotalus atrox (western diamondback rattlesnake) venom was studied for its specificity. HTb showed fibrinogenase activity, hydrolyzing the Aa chain of fibrinogen first, followed by the cleavage of the Bfl chain. HTb is different from thrombin and did not produce a fibrin clot. The degradation products of fibrinogen were found to be different, indicating that the cleavage sites in the Aa and B/~ chains are different from those of thrombin. N-Benzoyl-Phe-Val-Arg-p-nitroanUide was not hydrolyzed by HTb, although this substrate was hydrolyzed by thrombin and reptilase.

Snake venoms of the family Crotalidae and Viperidae contain a variety of proteolytic enzymes [1-4]. It was found by several investigators that venom proteinase activity was chelating agent dependent, suggesting the essential role of metal ions [5]. The presence of zinc was eventually reported by several workers [6-8]. When fibrinogen was reduced with mercaptoethanol [9], three constituent chains were obtained. The molecular weights were estimated as 63 800 for the Aa chain, 56 500 for the Bfl chain and 48 200 for the 7 chain (Fig. 1). When fibrinogen was incubated with the enzyme, there was a significant change in the appearance of the fibrinogen peak in analytical isotachophoresis (Fig. 2). After the digested fibrinogen had been reduced with mercaptoethanol, the three constituent polypeptide chains were separated by SDS-gel electrophoresis. The Aa chain was the first to be hydrolyzed, and the Aa band disappeared completely after 15 min. The fragment from the Aa chain had a molecular weight of about 39 800 and showed as a dark band

* Author to whom reprint requests should be addressed.

in the SDS-gel electrophoresis (Fig. 1, fl). The Bfl chain was also hydrolyzed by the enzyme, but it was more resistant to digestion than the Aa chain. The Bfl band in the SDS electrophoresis became weaker, and the fragment f2 (MW 27 800) (Fig. 1) was released. The 3' chain remained unchanged even after incubation with the enzyme for 120

"ff .

S

B

E --

120

LU

=E

60

I-

z _o

30

~

5

z_

c

Fig. 1. SDS gel electrophoresis of reduced fibrinogen after incubation with HTb. C, control (fibrinogen only); S, standard proteins. (a) Bovine serum albumin (66000); (b) ovalbumin (45000); (c) pepsin (34700); (d) trypsinogen (24000); (e) fllactoglobulin (18400); (f) lysozyme (14 300).

0167-4838/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

128 TABLE I THE EFFECT OF PURIFIED PROTEOLYTIC ENZYME HTb Substrate

Assay system

Effect of: crude venom

HTb

SDS-gel electrophoresis

n.d,

clot formation observation

n.d.

Aa degraded Bfl degraded no change no clot

N- Benzoyl-Phe-Val-Arg-p-nitroanilide (for thrombin and reptilase)

spectrophotometric

142.0 (nmol/mg per min)

Z-Phe-Arg-MCA (for kallikrein and crotalase)

fluorimetric fibrin plate method

73.8 (nmol/mg per min) lysis

18.0-10 3 (nmol/mg per min) no lysis

spectrophotometric

0

0

-butoxycarbonyl-L-alanine (for elastase)

spectrophotometric

0.05 (tLmol/mg per min)

0.03 (/~mol/mg per min)

360 (~t mol/mg per rain) (porcine pancreatic elastase)

Siccinyl-(Ala)3-p -nitroanilide (for elastase)

spectrophotometric

3.2 (nmol/mg per min)

2.7 nmol/mg per min

632 (nmol/mg per min) (porcine pancreatic elastase)

digestion of collagen gel in capillary tube

lysis

no lysis

fluorimetric

12.1.10 -3 (nmol/mg per min) n.d.

0

n.d.

+

0.22 (/~mol/mg per min)

2.4 (~ mol/mg per min)

Fibrinogen

Fibrin Congo red-elastin (for elastase)

purified enzyme (positive control)

p-Nitrophenyl-N-t-

Collagen gel (for collagenase) Succinyl-Gly-Pro-Leu-Gly-Pro-MCA (for collagenase)

+

Dimethylcasein

thin-layer and amino-acid analysis thin-layer and amino-acid analysis spectrophotometric

L-Leu-fl-naphthylamide (for leucine aminopeptidase)

spectrophotometric

6.7 (zaAss0/mg per min)

0

L-Leu-p-nitroanilide (for leucine aminopeptidase)

spectrophotometrie

2.0 (nmol/mg per min)

1.3 (nmol/mg per min)

Succinyl-Arg-Pro-Phe-His Leu-Leu-Val-Tyr-MCA (for renin)

fluorimetric

46.7.10 -3 (nmol/mg per min)

1.1.10 3 (nmol/mg per min)

Oxidized insurin B chain Glucagen

33.3 (nmol/mg per min) (porcine kidney leucine aminopeptidase)

129 TABLE I (continued) Effect of:

Assay system

Substrate

crude venom

N'~-p-Tosyl-L-arginine methyl ester (TAME)

spectrophotometric

280 (/~mol/mg per min)

N ~-Benzoyl-L-arginine ethyl ester (BAEE)

spectrophotometric

481 (~t mol/mg per min)

N-Acetyl-L-tyrosine ethyl ester (ATEE)

spectrophotometric

0

min. A coagulation test was done by mixing fibrinogen solution with the enzyme but no clot was observed. In normal blood coagulation, fibrinopeptides A and B are released by thrombin. The molecular weights of fibrinopeptides A and B are only 1900 and 2400, respectively; therefore, the remaining fragments must be very large and should have molecular weights around 62 000 and 54000. Our studies indicated that the molecular weights of the main fragments obtained were 39 800 for the Aa chain fragment and 27 800 for the BB chain fragment. This suggested that the HTb cleavage points in the Aa and BB chain were different from those in the case of thrombin. This view was compatible with the finding that a specific substrate for thrombin, N-benzoyl-Phe-Val-Arg-pnitroanilide was not hydrolyzed (Table I).

60

-~

~,o

~o N 90 100 Decreasing - ~

Decreasing

MOBILITY TOWARD ANODE

Fig. 2. lsotachophoresis indicating a change in the pattern of fibrinogen before (A) and after (B) incubating with HTb.

HTb

purified enzyme (positive control)

Bradykinin is released from bradykininogen by kallikrein and also by crotalase, a thrombin-like enzyme from C. atrox venom, but is not released by thrombin [3]. Kallikrein-like activity was tested using Z-Phe-Arg-MCA, but the activity of the purified enzyme was negligible compared with that of the crude venom. In order to determine whether fibrin was digested, the fibrin plate assay method was used. Crude venom was used as a positive control. Crude venom showed fibrinolytic activity, as a lysed area could be seen on a fibrin plate. However, no lysed region was observed when purified HTb was employed. For elastase activity determination, three substrates were used (Table I). No activity was observed when Congo red-elastin was used as a substrate. When two other synthetic substrates were used, a small amount of activity was detected, but it was barely more than background. For instance, with p-nitrophenyl-N-butoxycarbonyl-L-alanine, the activity was only 0.03 units, compared with 360 units for purified porcine pancreatic elastase, or only 0.008% of the activity of porcine pancreatic elastase. Similarly,. the activity was only 0.43% of that of porcine pancreatic elastase when succinyl-(Ala)3-p-nitroanilide was used as the substrate. Collagenase activity was determined by the lysis of collagen gel in a capillary tube and by a fluorimetric method using succinyl-Gly-Pro-LeuGIy-Pro-MCA as a substrate. No activity was detected with either method, although such activity could be seen with crude venom. This result was further substantiated by the immunodiffusion

130

- - 3.0

A

.E E

>"1~ i- E

"0

- - o 1.0 OE uJ~

o.g I

i

20 40 60 TEMPERATURE

: 20

I

80 I°C I

B

creatic chymotrypsin. The substrate acetyltyrosine ethyl ester was not hydrolyzed by the enzyme. It was also found that the purified enzyme does not possess leucine aminopeptidase and renin activities (Table I). The effect of temperature on the proteolytic activity was investigated using dimethylcasein as a substrate. As illustrated in Fig. 3, the enzyme had an optimum activity around 42°C. The proteolytic activity was almost completely lost above 80°C. The ascending portion of Fig. 3A (0 to 42°C) was replotted as log V vs. 1 / T (Fig. 3B), where V is the reaction velocity and T is the temperature in kelvin. From the Arrhenius plot, the activation energy was found to be 6390 c a l / m o l for HTb.

1.6

References 1.2

o 0[ ;

T

35o 3,o 1/T [°C-1 ]

37o x10-5

Fig. 3. (A) The effect of temperature on the proteolytic activity ofthe enzyme with dimethylcasein as a substrate. (B) The Arrhenius plot for the determination of the activation energy.

study, in which precipitin lines with anticollagenase (from C. atrox) were observed for crude venom, but no such line was observed for purified enzyme. Both tosylarginine methyl ester and benzoylarginine ethyl ester are commonly used for trypsin activity assays. Neither of these substrates was hydrolyzed by the purified enzyme. The purified enzyme is not the same as pan-

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