[37]

SNAKE VENOM PROTEINASES

[37] P r o t e i n a s e s

Agkistrodon

459

from the Venom of

halys blomhoffii

By SADAAKI IWANAGA, GENICHmO OSHI~A, and TOMOJI SUZUKI Snake venoms contain several types of proteinases including en'dopeptidases, nonproteolytic arginine ester hydrolases, thrombinlike enzymes and kininogenase. 1-4 The venom endopeptidase is mainly distributed in families of Crotalidae and Viperidae. A common feature of this proteinase from the Crotalidae venoms is that they all hydrolyze peptide bonds with amino groups contributed by leucine and phenylalanine residues, and require more than a hexapeptide sequence for hydrolysis of this bond. Another common characteristic point is that they are easily inactivated by E D T A and cystcine. In these properties, they differ from well known mammalian endopeptidase.

Assay Method 5

Principle. The enzyme is assayed with 2.0% casein solution in TrisHC1 buffer, pH 8.5, at 37 °. Undigested casein is precipitated with trichloroacetic acid and filtered or centrifuged. Digested casein in the supernatant is determined with the Folin-Ciocalteau reagent. Reagents Tris-HC1 buffer, 0.4 M, pH 8.5 Casein, 2.0%: Two grams of casein (Hammarsten quality) are suspended in 100 ml of 0.4 M Tris-HC1 buffer, pH 8.5 and heated for 15 min, in a boiling water bath to make complete solution. Trichloroacetic acid (TCA), 0.44 M Sodium carbonate, 0.4 M Folin-Ciocalteau reagent. The commercially available reagen~ is diluted three times with distilled water before use. Standard tyrosine solution: The tyrosine standard is prepared by dissolving 18.10 mg of pure, dry tyrosine in 1 liter of 0.2 M HC1. E. Kaiser and H. Michl, "Die Biochemie der tierischen Gifte." Deuticke, Vienna, 1958. '"Venomous Animals and Their Venoms" (W. Bucherl, E. E. Buckley, and V. Deulofeu, eds.), Vol. 1. Academic Press, New York, 1968. O. B. Henriques and S. B. Henriques, "Pharmacology and Toxicology of Naturally Occurring Toxins," Vol. 1, p. 215. Pergamon, Oxford, 1971. 4 H. F. Deutsch and C. R. Diniz, J. Biol. Chem. 216, 17 (1955). Y. Murata, S. Satake, and T. Suzuki, J. Biochem. 53, 431 (1963).

460

ENDOPEPTIDASES

[37]

Procedure. Casein solution 0.5 ml, is digested for 15 min at 37 ° with 0.5 ml of a suitably diluted enzyme solution. The reaction is terminated by adding 1.0 ml of 0.44 M TCA; after it has stood for 30 min, the mixture is filtered through Toyo filter paper No. 2 or Whatman filter paper No. 2. To 0.5 ml of filtrate, 2.5 ml of 0.4 M Na2C03, and 0.5 ml of Folin-Ciocalteau reagent are added. After the preparation has stood for 20 min, the blue color is read at 660 nm and compared against a standard tyrosine solution. Definition o/ Unit and Specific Activity2 One unit of proteinase, expressed as proteinase units (PU) is defined as the amount of enzyme yielding an increase in color equivalent to 1.0 ~g of tyrosine per minute with Folin's reagent. Specific activity is expressed as units per milligram of protein. P u r i f i c a t i o n P r o c e d u r e 6-9

The following procedures are conducted at 4°-6 °.

Step 1. Chromatographic Separation of Proteinases/ A sample of 3 g (total absorbance at 280 nm = 3600) of lyophilized Agkistrodon halys blomhoffii venom is dissolved in 10 ml of 0.005 M sodium acetate and applied to a column of DEAE-cellulose (Serva, 0.68 meq/g, 2.5 )~ 70 cm) equilibrated with 0.005 M sodium acetate, pH 7.0. The exponential gradient elution is started with 1 liter of the equilibration buffer in the mixing vessel and 0.1 M sodium acetate, pH 7.0, in the reservoir. The latter is replaced by 0.2 M and 0.5 M sodium acetate, pH 7.0, as indicated in Fig. 1. The flow rate is adjusted to about 30 ml/hr, and the eluate is collected in 10-ml fractions. Figure 1 shows a typical elution profile. The proteinase activities are separated into three peaks, which are designated in the order of their elution from the column as proteinases a, b, and c. This pattern is very reproducible, always giving three proteolytic peaks. Proteinase a is found in the first peak in which the proteins are not adsorbed by DEAE-cellulose. Proteinase b, however, emerges occasionally a little faster or later than the associating peak of protein. Proteinase c is demonstrated always as the last peak of protein. Because the content of proteinase b is highest among the three proteinases in this venom, amounting approximately 10.4% of the lyophilized venom by weight, the following purification procedure is described on e S. Satake, Y. Murata, and T. Suzuki, J. Biochem. 53, 438 (1963). 7 S. Iwanaga, T. Omori, G. Oshima, and T. Suzuki, J. Biochem. 57, 392 (1965). 8 G. Oshima, S. Iwanaga, and T. Suzuki, J. Biochem. 64, 215 (1968). G. Oshima, Y. Matsuo, S. Iwanaga, and T. Suzuki, J. Biochem. 64, 227 (1968).

[37]

SNAKE VENOM P R O T E I N A S E S

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I

I

I

l

to 0.1 M

0.005M

I

~0 0 . 2 M

461 I

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2.0

I

a

30

b

A ti 2 5 -

1.5

20w

I.O 15-

ofj

I0-

0.5

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5-

40

80

120

180

200

24O

28O

32O

TUBE NUMBER

FIa. 1. Separation of three proteinases, a, b, and c, from the venom of Agkistrodon halys blomho/]ii by column chromatography on DEAE-cellulose. Experimental conditions: column, 2.5 X 70 cm; sample volume, 3 g/10 ml; flow rate, 30 ml/hr; fraction volume, 10 ml per tube; exponential gradient, 0.005 M to 0.1 M, 0.2 M and 0.5 M sodium acetate as indicated. Reproduced from S. Iwanaga, T. Omori, G. Oshima, and T. Suzuki, J. Biochem. 57, 392 (1965). this enzyme. Further purification methods of proteinases a and c are described in the literatures2 ,~ Step 2. Gel Filtration of Proteinase b Fraction on a Sephadex G-tO0 Column2 The fractions (tubes 190 to 250) with proteinase b activity eluted with 0.2 M acetate are combined and concentrated to a half volume of the pooled fraction by lyophilization. The concentrate is dialyzed against 2 liters of distilled water for 24 hr with two changes, and the dialyzed solution is then freeze-dried. The proteinase b fraction (total absorbance at 280 nm = 2530), which was prepared from 32.7 g of the unfractionated venom, is dissolved in 26.3 ml of 0.05 M sodium acetate, pH 7.0. The solution is applied to a column of Sephadex G-100 (6.5 X 866 cm), previously equilibrated with the same buffer and eluted with the same buffer at a flow rate of about 100 ml/hr. Fractions of 15 ml per tube are collected. Figure 2 shows the elution profile. The proteins are eluted in three peaks, and the first peak with proteolytic activity is concentrated by lyophilization. The concentrate is desalted on a column of Sephadex G-25 (6.5 X 60 cm) in distilled water and lyophilized.

462

137]

ENDOPEPTIDASES J

E

A

4.0

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Proteinases from the venom of Agkistrodon halys blomhoffi.

[37] SNAKE VENOM PROTEINASES [37] P r o t e i n a s e s Agkistrodon 459 from the Venom of halys blomhoffii By SADAAKI IWANAGA, GENICHmO OSHI~A,...
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