874
NATURALLY OCCURRING PROTEASE INHIBITORS
[78]
AMINO ACID COMPOSITIONS OF THE BRONCHIAL MUCUS INHIBITORS (BSI) a
Residue
BSI-I (moles/mole)
BSI-II (%)
Asp Thr Ser Glu Pro Gly Ala Cysll2 Val Met Ile Leu Tyr Phe Lys His Arg
16 9 15 12 12 6 5 12 2 2 6 6 3 3 8 4 4
13.9 6.3 4.1 8.4 9.4 4.8 3.8 8.1 3.4 5.8 3.8 6.2 5.3 3.9 6.3 2.1 4.2
The results are calculated from 24, 48, and 72 hr hydrolysis.
The amino acid compositions of the inhibitors are given in the table. Acknowledgment This work was supported by Sonderforschungsbereich 51, Munich, of the Deutsche Forschungsgemeinschaft.
[78] P r o t e i n a s e I n h i b i t o r s f r o m t h e V e n o m o f Russell's V i p e r B y SADAAKI IWANAGA, HIDENOBU TAKAHASHI, a n d TOMOJI SUZUKI
Snake venom contains a number of physiologically active polypeptides and peptides, including neurotoxin, cardiotoxin, cytotoxin, and kinin potentiator and others, and the chemical structures of some of them have been established. 1-3 Recently, a new class of polypeptides, 1A. T. Tu, Annu. Rev. Biochem. 42, 235 (1973). 2 E. Karlsson, Experientia 29, 1319 (1973). L. Ryden, D. Cabel, and D. Eaker, Int. J. Peptide Protein Res. 5, 261 (1973).
[78]
PROTEINASE INHIBITORS FROM RUSSELL'S VIPER
875
which inhibits proteolytic activities of kallikrein, plasmin, trypsin, and a-chymotrypsin, has been found in several snake venoms. 4 These proteinase inhibitors are mainly distributed in the venoms of members of the Viperidae and Elapidae, 5 and the inhibitors isolated from the venom of Russell's viper (RVV), Hemachatus haemachatus ( H H V ) , and N a j a nivea (NNV) are a basic polypeptide with a molecular weight of about 6500. 6-8 Assay M e t h o d Principle. The hydrolysis of N~-toluenesulfonyl-L-arginine methyl ester (TAME) by trypsin is measured by the hydroxamate method of Robert. ° Reagents
Tris-HCl buffer, 0.4 M, pH 8.5 Substrate solution: 0.1 M T A M E (378.9 mg of T A M E . H C 1 are dissolved in 10 ml of distilled water and stored at 4 °) Enzyme. Three times recrystallized bovine trypsin (Worthington Biochemical Corp., Freehold, New Jersey) is dissolved in 0.001 M HC1 to give a final concentration of 2 mg/ml and stored at 0 °. The enzyme solution is diluted to five times with the same hydrochloric acid before use. Alkaline hydroxylamine solution: 2 M (13.9%) aqueous hydroxylamine hydrochloride is mixed with an equal volume of 3.5 M N a O H just before use. Store 2 M hydroxylamine at 4 °. Trichloroacetic acid (TCA) solution: 6 g of TCA dissolved in 100 ml of 3 M HC1 Ferric chloride solution: 14.87 g of FeCl~.6H~O dissolved in 500 ml of 0.04 N HC1 and stored in a dark bottle Snake venom: Lyophilized or desiccated venoms were dissolved in 0.15 M NaC1 to give a concentration of 1.0 mg/ml, and the solutions were heated at 80 ° for 10 rain to inactivate proteolytie enzymes contained in the venom. Precipitates appearing after heat treatment were removed by centrifugation at 5000 rpm for 10 min. 4It. Takahashi, S. Iwanaga, and T. Suzuki, FEBS Lett. 27, 207 (1972). H. Takahashi, S. Iwanaga, and T. Suzuki, Toxicon 12, 193 (1974). ° H. Takahashi, S. Iwanaga, and T. Suzuki, J. Biochem. 76, 709 (1974). H. Takahashi, S. Iwanaga, Y. Hokama, T. Suzuki, and T. Kitagawa, FEBS Lett. 38, 217 (1974). H. Takahashi, S Iwanaga, T. Kitagawa, Y. Hokama, and T. Suzuki, J. Biochem. 76, 721 (1974). Dp. S. Robert, J. Biol. Chem. 232, 285 (1958).
876
NATURALLY
OCCURRING PROTEASE INHIBITORS
[78]
Procedure. For the determination of inhibitory activity, a mixture of 0.1 ml of trypsin solution (4 t~g) and unfractionated venom or the fraction containing venom proteinase inhibitor in 0.9 ml of 0.4 M Tris-HC1 buffer, pH 8.5, is incubated for 10 min; then 0.1 ml of 0.1 M TAME is added. After 30 min, the reaction is terminated by the addition of 0.5 ml of 6% TCA, and then 1.0 ml of alkaline hydroxylamine solution is added. (If a precipitate appears after addition of TCA, the preparation is allowed to stand at room temperature for at least 30 min, and the precipitate is removed by filtration or centrifugation; the filtrate or supernatant is mixed with the hydroxylamine solution.) After the mixture has stood at room temperature for 30 min, 2.0 ml of ferric chloride solution is added; the color developed is measured in 30 min at 500 nm. Formation of gas bubbles in the colorimeter cell is largely prevented if the solution is read against a reagent blank prepared by substituting buffer for enzyme and TAME. A blank of TAME should also be included, which is prepared by substituting buffer for enzyme. Definition o] Unit. One unit is defined as the amount causing reduction of TAME hydrolysis by 1 ~mole/min2 Purification P r o c e d u r e 4,6
Step 1. Gel filtration on Sephadex G-75. Lyophilized venom of Russell's viper (100 mg, lot $62B-206, obtained from Sigma Chemical Co., St. Louis, Missouri) is dissolved in 5 ml of 0.04 M Tris-HC1 buffer, pH 8.5, containing 0.1 M NaCl, and the solution is applied to a column of Sephadex G-75 (2.0 X 128 cm) equilibrated with the same buffer. The column is eluted with the equilibration buffer at 4 ° and fractions of 5 ml are collected at a flow rate of 50 ml/hr. Figure 1 shows a typical elution pattern. The E peak contains proteinase inhibitors, and the fractions indicated by a solid bar are combined and lyophilized. The dried material (about 32 mg) is dissolved in 2 ml of distilled water and applied to a column (2.5 }( 94 cm) of Sephadex G-50 equilibrated with 0.1 M (NH4) HCO3, pH 8.2, to remove Tris-HC1 buffer. The resulting unvolatile buffer-free fractions containing the inhibitor are combined, lyophilized, and sublimated at 40 ° . Step 2. Chromatography on SE-Sephadex C-25. The dried material (15 mg) of step 1 is dissolved in 2 ml of 0.04 ammonium formate buffer, pH 4.0, and applied to a column of SE-Sephadex C-25 (2.0 X 30 cm) equilibrated with the same buffer. Linear gradient elution is started with 500 ml of each of the equilibration buffer in the mixing vessel and 0.2 M ammonium acetate, pH 9.5, in the reservoir. Then, the column is eluted with a gradient, formed with 0.2 M buffer to 0.5 M, pH 9.5, to elute all
[78]
PROTEINASE INHIBITORS FROM RUSSELL'S VIPER
877
0.7
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0.2
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FRACTION NUMBER (Sml/tube)
Fla. 1. Gel filtration of Russell's viper venom on a Sephadex G-75 column (2.5 X 128 cm). Experimental conditions: sample volume, 5 ml per 100 mg of crude venom; elution buffer, 0.04 M Tris-HC1 buffer, pH 8.5, containing 0.1 M NaC1; flow rate, 50 ml/hr; fraction volume, 5 ml/tube. The fractions indicated by the solid bar were collected and lyophilized. Reproduced from H. Takahashi, S. Iwanaga, and T. Suzuki, I. Biochem. 76, 711 (1974) the adsorbed materials. Fractions of 5 ml are collected at 4 ° at a flow rate of 30 ml/hr. Figure 2 shows the elution pattern. Inhibitory activities were found in two peaks, and these fractions were combined and lyophilized (Fig. 2). Ammonium acetate is removed from the dried material in vacuo (0.005 mm Hg) at 40 °. The overall yields of RVV ~nhibitor I and I I are about 1 and 5 mg, respectively, from 100 mg of crude venom. The above procedures are reproducible and are applicable also for the isolation of proteinase inhibitors from the venoms of H e m a c h a t u s haemachatus 1° and N a j a nivea. ~ Properties 4,6 Purity. Both RVV inhibitor I and I I give a single band on disc polyacrylamide-gel electrophoresis in the presence and in the absence of sodium dodecyl sulfate (SDS). Physical' Properties. The molecular weights of both inhibitors I and I I are about 7200 (___1000), as estimated with SDS gel (7.5%) electro10H. Takahashi, S. Iwanaga, T. Kitagawa, Y. Hokama, and T. Suzuki, Proteinase Inhibitor, Proc. Int. Res. Con]., 2nd (Bayer Symp. V), Grosse Ledder, 1973, p. 265. 11T. Tatsuki, Y. Hokama, S. Iwanaga, and T. Suzuki, Seikagaku 46, 704 (1974). In Japanese.
878
NATURALLY OCCURRING PROTEASE INHIBITORS
1T ~.=.=.=
.=.= .=-=---x---=---N
---k---x---
x---
=.A-= "=
i
X---~---K---. .-'°
2
O.I
Io
J
0
0
~
200 FRACTION
300
[78]
u~ 20
I0
" I0
5
Z
I--. Z
_o
0
0
~
'
400
NUMBF'R f .~ml I h,h-t
FIG. 2. Purification of the Russell's viper venom proteinase inhibitor on a SESephadex column (2.0 × 30 cm). Experimental conditions: sample, the pooled fraction (15 mg) of Fig. 1; flow rate, 35 ml/hr; fraction volume, 5 ml/tube. A linear gradient elution starts with 500 ml each of 0.04 M ammonium formate buffer, pH 4.0, and 0.2 M ammonium acetate buffer, pH 9.5, and then a gradient of 0.2 M to 0.5 M buffer, pH 9.5, is used to elute all adsorbed material. Reproduced from H. Takahashi, S. Iwanaga, and T. Suzuki, F E B S Lett. 27, 207 (1972).
phoresis. The ultraviolet absorption spectra of these inhibitors are identical and show an absorption maximum at 278 nm and minimum at 256 nm at pH 9.0. The extinction coefficients of 1.0% polypeptide solutions of inhibitors I and II at 280 nm are 5.3 and 5.5, respectively. Stability. RVV inhibitors I and II are stable in the frozen and lyophilized state for at least one year and no loss of inhibitory activity is observed when inhibitor II is heated at pH 2 to 7.5 at 90 ° for 15 min. However, above pH 8.0 it is denatured. Specificity. The following enzymes are strongly inhibited by RVV inhibitors I and II: plasma and pancreatic kallikreins, trypsin, a-chymotrypsin, and plasmin from bovine source and human plasmin. The inhibitor II does not inhibit human and bovine thrombins, snake venom kininogenase, Reptilase, bromelain, ficin, papain, subtilisin BPN', thermolysin, and carboxypeptidases A and B. Kinetic Properties. RVV inhibitors I and II inactivate bovine trypsin, probably by formation an enzyme-inhibitor complex in a molar ratio of 1:1. The Ki values of inhibitor II, measured using synthetic ester substrate (TAME), are 0.76 nM for bovine trypsin, 0.14 nM (N ~acetyltyrosine ethyl ester) for bovine a-chymotrypsin; 0.29 nM for bovine plasma kallikrein, and 1.0 nM for bovine plasmin.
[78 !
879
PROTEINASE INHIBITORS FROM RUSSELL'S VIPER
TABLE I AMINO ACID COMPOSITIONSa OF PROTEINASE INHIBITORS ISOLATED FROM THE VENOMS OF RUSSELL'S VIPER, Hemachatus haemachatus, AND Naja nivea
Amino acid Lys His Ammonia Arg Asp Thr Ser Glu Pro Gly Ala Cys112 Val Met Ile Leu Tyr Phe Trp
Russell's viper, II
Russell's viper, I
3 2 (12) 7 8 3 2 5 2 8 2 6 1 0 1 3 3 4 0 60
2 2 (11) 5 7 2 2 5 5 7 3 4 1 0 2 1 1 3 0 52
H. haema- N. nivea chatus II II 2 1 (7) 5 5 3 1 6 2 6 5 6 1 0 3 4 3 4 0 57
2 2 (6) 7 4 3 1 6 2 6 5 6 1 0 3 3 2 4 -57
N. nwea Ia
N. nwea Ib
2 2 (7) 6 4 3 1 7 2 6 5 6 1 0 3 3 2 4 -57
4 1 (6) 4 5 3 2 4 3 3 4 6 3 1 3 3 2 3 -54
a The compositions were calculated (in residues per mole) by extrapolation or from the average values estimated on samples after 24-, 48-, and 72-hr hydrolyzates.
A m i n o Acid C o m p o s i t i o n 2 ,1° T h e c o m p o s i t i o n s of s e v e r a l i n h i b i t o r s i s o l a t e d f r o m t h e v e n o m s of R u s s e l l ' s v i p e r , H e m a c h a t u s haemachatus, a n d N a j a nivea a r e s h o w n in T a b l e I. C o m m o n f e a t u r e s i n c l u d e r e l a t i v e l y h i g h c y s t i n e c o n t e n t a n d f e w or no m e t h i o n i n e a n d t r y p t o p h a n r e s i d u e s ; no s u g a r r e s i d u e is d e t e c t e d in a n y i n h i b i t o r . A m i n o Acid Sequence. 7,s T h e s e q u e n c e s of R V V i n h i b i t o r I I a n d H H V i n h i b i t o r I I a r e s h o w n in F i g . 3, in c o m p a r i s o n w i t h t h o s e of b o v i n e p a n c r e a t i c t r y p s i n i n h i b i t o r 1~ a n d s n a i l i s o i n h i b i t o r K. 18 T h e o v e r a l l p r i m a r y s t r u c t u r e of s n a k e v e n o m p r o t e i n a s e i n h i b i t o r s is q u i t e s i m i l a r to t h o s e of t h e K u n i t z - t y p e i n h i b i t o r s , i n d i c a t i n g 5 0 - 6 0 % s e q u e n c e h o m o l o g i e s . M o r e o v e r , t h e 6 h a l f - c y s t i n e r e s i d u e s of t h e s e f o u r i n h i b i t o r s a r e in t h e s a m e p o s i t i o n s a l o n g t h e p o l y p e p t i d e s (Fig. 4). T h e r e a c t i v e sites of ~2B. Kassell, M. Radicevic, M. J. Anfield, and M. Laskowski, St., Biochem. Biophys. Res. Commun. 18, 255 (1965). ~3H. Tschesche, Angew. Chem. 86, 21 (1974).
880 RVV II HHV If SNAIL IK BPTI
NATURALLY OCCURRING PROTEASE INI-IIBITORS
[78]
I 0 15 ~ 20 Hls-Asp-Arg-Pco-TIP-Phe-Cys-Asn-Leu-Ala-Pro-G]u-Ser-G}y-Arg-Cys-Arg-G y-Hts-LeuArg-Pro-Asp-Phe-Cys-G]u-Leu-Pro-Ala-Glu-Thr-Gly-Leu-Cys-Lys-Ala-Tyr-liePyr-••y-A•g-P••-Asp-Phe-Cys-G]u-Leu-Pr•-A•a-••u-Th•-••y-P••-Cys-Lys-A•a-Ser-PheArg-Pro-Asp-Phe-Cys-Leu-G]u-Pro-Pro-Tyr-Thr-Giy-Pro-Cys-Lys-Ala-Arg-I]e-
5 30 5 40 Arg-Ar~-~e-Tyr~yr~Asn-Leu~B~u-Ser-Asn~Lys-~ys~Lys~a~-P~e-Phe-Tyr-G~y-G~y-CysArg-Ser-Phe-•is-Tyr-Asn-Leu-A•a-A•a-G•n-Lys-Cys-Leu-G]u•Phe-••e-Tyr-G•y-••y-CysAr~-G~n~Tyr-T~r-Tyr-Asn-Ser-Lys~Ser-~y-G~y-Cys-~n-G~n-Phe-~e-Tyr-G~y-~y-Cys~e-Arg-Tyr~Phe-Tyr-Asn-A~a-Lys-A~a-~y-Leu-Cys-~n-Thr-Phe-Va~-Tyr-G~y-~y-Cys45 50 5 G~y-G~y-Asn~A]a-Asn-Asn~Phe-G~u-Thr.~Arg-Asp-G~u-Cys-Ar~-G~u-Thr-Cys~G~y-G]y-L~ Giy-G]y-Asn-A]a-Asn-Arg-Phe-Lys-Thr-Ile-Asp-Glu-Cys-Arg-Arg-Thr-Cys-Va]-Gly Arg-Gly-Asn-Gln-Asn-Arg-phe-Asp-Thr-Thr-G]n-G]n-Cys-G]n-Gly-Va]-Cys-Yal
Arg-kla-Lys-Arg-Asn-Asn-Phe-Lys-Ser-Ala-Glu-Asp-Cys-Met-Arg-Thr-Cys-Gly-Gly-Ala FIG. 3. Comparison of the amino acid sequences of Russell's viper venom (RVV) inhibitor II, Hemachatus haemachatus (HHV) inhibitor II, snail isoinhibitor K, and bovine pancreatic trypsin inhibitor (Kunitz type). Based on H. Takahashi, S. Iwanaga, Y. tIokama, T. Suzuki, and T. Kitagawa, FEBS Left. 38, 217 (1974). B. Kassell, M. Radicevic, M. J. Anfield, and M. Laskowski, Sr., Bioehem. Biophys. Res. Commun. 18, 255 (1965) ; and H. Tschesche, Angew. Chem. 86, 21 (1974).
RVV inhibitor II and H H V inhibitor II are still unknown, but the most probable site for interaction with trypsin is the peptide linkage ArglTGlyl+ in the former, and Lysls-Ala~6 in the latter, because the homologies of all the inhibitors around these portions are extremely high. These results are very interesting in relation to the evolution of these inhibitors, since evolutionary histories of cows, snakes, and snails are quite different. I
i
FIG. 4. A schematic representation of the structure of the proteinase inhibitor isolated from the venom of Russell's viper. Based on T. Takahashi, S. Iwanaga, Y. Hokama, T. Suzuki, and T. Kitagawa, FEBS Let$. 38, 217 (1974) ; and H. Takahashi, S. Iwanaga, T. Kitagawa, Y. Hokama, and T. Suzuki, J. Biochem. 76, 721 (1974).
[79]
BROAD-SPECIFICITY INHIBITORS FROM SEA ANEMONES
881
TABLE II DISTRIBUTION OF PROTEINASE INHIBITORS IN VARIOUS SNAKE VENOMS a'b
Family
Bovine plasma kallikrein
Bovine plasma plasmin
Bovine pancreatic trypsin
Bovine pancreatic (~-chymotrypsin
12 40 88
18 91 110
5 22 74
5 77 217
100 100 100 4 125 215
62 12 42 20 34 43
55 3 60 22 18 28
80 15 20 13 34 105
Viperidae
V. russelli V. ammodytes B. arietans Elapidae
N. N. N. H. D. D.
hannah nivea haje haemachalus angusticeps polylepis
a From H. Takahashi, S. Iwanaga, a n d T. Suzuki, Toxicon 12, 193 (1974). b The values were expressed as the quantities (#g) to show 50 % inhibition of the activity of the proteinases. The following venoms showed no inhibitory activity when 100 t~g of each was used: V. pale~tinae, E. carinatu~, B. gabon@a, N. naja
atra, N. melanoleuca, N. nigricollzs, N. naja samarens~s, N. naja, B. fasczatus, B. mult~cinctus, L. semifasciata, L. lahcaudata, A. halys blomho~,, A. acutus, A. rhodostoma, A. p~scwo~us leucostoma, A. contortmx contortr~x, A. p~scivorus piscworus, A. contortr~x mokeson, C. atrox, C. adamanteus, C. viridis wr~dis, C. basiliscus, C. durissus terrificus, B. atrox, B. jararaca, T. flavoviridis, T. gramineus, T. okinavensis, T. mucrosquamalus, Causus rhombeatus.
Distribution2 ,14 Proteinase inhibitor of the same type as described here is demonstrated also in the venoms of several snakes of the Elapidae and Viperidac (Table II). However, it is not found in Crotalidae and Hydrophiidae venoms. ~4D. J. Strydom, Nature (London) New Biol. 243, 88 (1973).
[79] B r o a d - S p e c i f i c i t y I n h i b i t o r s f r o m Sea Anemones By GERT WUNDERER, L~SZLO BI~RESS, WERNER MACHLEIDT, and HANS FRITZ In the purification of neurotoxins from Anemonia sulcata, fractions with antitryptic activity were observed. 1 This inhibitory activity could be ascribed to a variety of basic polypeptides. ~,~ The broad specificity 1 L. B6ress and R. B~ress, Kiel. Meeres]orsch. 27, 117 (1971). : H. Fritz, B. Brey, and L. B6ress, Hoppe-Seyler's Z. Physiol. Chem. 353, 19 (1972). 3G. Wunderer, K. Kummer, H. Fritz, L. B~ress, and W. Machleidt, Proteinase Inhibitors, Proc. Int. Res. Con]., 2nd (Bayer Symp. V), Grosse Ledder, 1973, p. 277.
NATURALLY OCCURRING PROTEASE INHIBITORS
[78]
AMINO ACID COMPOSITIONS OF THE BRONCHIAL MUCUS INHIBITORS (BSI) a
Residue
BSI-I (moles/mole)
BSI-II (%)
Asp Thr Ser Glu Pro Gly Ala Cysll2 Val Met Ile Leu Tyr Phe Lys His Arg
16 9 15 12 12 6 5 12 2 2 6 6 3 3 8 4 4
13.9 6.3 4.1 8.4 9.4 4.8 3.8 8.1 3.4 5.8 3.8 6.2 5.3 3.9 6.3 2.1 4.2
The results are calculated from 24, 48, and 72 hr hydrolysis.
The amino acid compositions of the inhibitors are given in the table. Acknowledgment This work was supported by Sonderforschungsbereich 51, Munich, of the Deutsche Forschungsgemeinschaft.
[78] P r o t e i n a s e I n h i b i t o r s f r o m t h e V e n o m o f Russell's V i p e r B y SADAAKI IWANAGA, HIDENOBU TAKAHASHI, a n d TOMOJI SUZUKI
Snake venom contains a number of physiologically active polypeptides and peptides, including neurotoxin, cardiotoxin, cytotoxin, and kinin potentiator and others, and the chemical structures of some of them have been established. 1-3 Recently, a new class of polypeptides, 1A. T. Tu, Annu. Rev. Biochem. 42, 235 (1973). 2 E. Karlsson, Experientia 29, 1319 (1973). L. Ryden, D. Cabel, and D. Eaker, Int. J. Peptide Protein Res. 5, 261 (1973).
[78]
PROTEINASE INHIBITORS FROM RUSSELL'S VIPER
875
which inhibits proteolytic activities of kallikrein, plasmin, trypsin, and a-chymotrypsin, has been found in several snake venoms. 4 These proteinase inhibitors are mainly distributed in the venoms of members of the Viperidae and Elapidae, 5 and the inhibitors isolated from the venom of Russell's viper (RVV), Hemachatus haemachatus ( H H V ) , and N a j a nivea (NNV) are a basic polypeptide with a molecular weight of about 6500. 6-8 Assay M e t h o d Principle. The hydrolysis of N~-toluenesulfonyl-L-arginine methyl ester (TAME) by trypsin is measured by the hydroxamate method of Robert. ° Reagents
Tris-HCl buffer, 0.4 M, pH 8.5 Substrate solution: 0.1 M T A M E (378.9 mg of T A M E . H C 1 are dissolved in 10 ml of distilled water and stored at 4 °) Enzyme. Three times recrystallized bovine trypsin (Worthington Biochemical Corp., Freehold, New Jersey) is dissolved in 0.001 M HC1 to give a final concentration of 2 mg/ml and stored at 0 °. The enzyme solution is diluted to five times with the same hydrochloric acid before use. Alkaline hydroxylamine solution: 2 M (13.9%) aqueous hydroxylamine hydrochloride is mixed with an equal volume of 3.5 M N a O H just before use. Store 2 M hydroxylamine at 4 °. Trichloroacetic acid (TCA) solution: 6 g of TCA dissolved in 100 ml of 3 M HC1 Ferric chloride solution: 14.87 g of FeCl~.6H~O dissolved in 500 ml of 0.04 N HC1 and stored in a dark bottle Snake venom: Lyophilized or desiccated venoms were dissolved in 0.15 M NaC1 to give a concentration of 1.0 mg/ml, and the solutions were heated at 80 ° for 10 rain to inactivate proteolytie enzymes contained in the venom. Precipitates appearing after heat treatment were removed by centrifugation at 5000 rpm for 10 min. 4It. Takahashi, S. Iwanaga, and T. Suzuki, FEBS Lett. 27, 207 (1972). H. Takahashi, S. Iwanaga, and T. Suzuki, Toxicon 12, 193 (1974). ° H. Takahashi, S. Iwanaga, and T. Suzuki, J. Biochem. 76, 709 (1974). H. Takahashi, S. Iwanaga, Y. Hokama, T. Suzuki, and T. Kitagawa, FEBS Lett. 38, 217 (1974). H. Takahashi, S Iwanaga, T. Kitagawa, Y. Hokama, and T. Suzuki, J. Biochem. 76, 721 (1974). Dp. S. Robert, J. Biol. Chem. 232, 285 (1958).
876
NATURALLY
OCCURRING PROTEASE INHIBITORS
[78]
Procedure. For the determination of inhibitory activity, a mixture of 0.1 ml of trypsin solution (4 t~g) and unfractionated venom or the fraction containing venom proteinase inhibitor in 0.9 ml of 0.4 M Tris-HC1 buffer, pH 8.5, is incubated for 10 min; then 0.1 ml of 0.1 M TAME is added. After 30 min, the reaction is terminated by the addition of 0.5 ml of 6% TCA, and then 1.0 ml of alkaline hydroxylamine solution is added. (If a precipitate appears after addition of TCA, the preparation is allowed to stand at room temperature for at least 30 min, and the precipitate is removed by filtration or centrifugation; the filtrate or supernatant is mixed with the hydroxylamine solution.) After the mixture has stood at room temperature for 30 min, 2.0 ml of ferric chloride solution is added; the color developed is measured in 30 min at 500 nm. Formation of gas bubbles in the colorimeter cell is largely prevented if the solution is read against a reagent blank prepared by substituting buffer for enzyme and TAME. A blank of TAME should also be included, which is prepared by substituting buffer for enzyme. Definition o] Unit. One unit is defined as the amount causing reduction of TAME hydrolysis by 1 ~mole/min2 Purification P r o c e d u r e 4,6
Step 1. Gel filtration on Sephadex G-75. Lyophilized venom of Russell's viper (100 mg, lot $62B-206, obtained from Sigma Chemical Co., St. Louis, Missouri) is dissolved in 5 ml of 0.04 M Tris-HC1 buffer, pH 8.5, containing 0.1 M NaCl, and the solution is applied to a column of Sephadex G-75 (2.0 X 128 cm) equilibrated with the same buffer. The column is eluted with the equilibration buffer at 4 ° and fractions of 5 ml are collected at a flow rate of 50 ml/hr. Figure 1 shows a typical elution pattern. The E peak contains proteinase inhibitors, and the fractions indicated by a solid bar are combined and lyophilized. The dried material (about 32 mg) is dissolved in 2 ml of distilled water and applied to a column (2.5 }( 94 cm) of Sephadex G-50 equilibrated with 0.1 M (NH4) HCO3, pH 8.2, to remove Tris-HC1 buffer. The resulting unvolatile buffer-free fractions containing the inhibitor are combined, lyophilized, and sublimated at 40 ° . Step 2. Chromatography on SE-Sephadex C-25. The dried material (15 mg) of step 1 is dissolved in 2 ml of 0.04 ammonium formate buffer, pH 4.0, and applied to a column of SE-Sephadex C-25 (2.0 X 30 cm) equilibrated with the same buffer. Linear gradient elution is started with 500 ml of each of the equilibration buffer in the mixing vessel and 0.2 M ammonium acetate, pH 9.5, in the reservoir. Then, the column is eluted with a gradient, formed with 0.2 M buffer to 0.5 M, pH 9.5, to elute all
[78]
PROTEINASE INHIBITORS FROM RUSSELL'S VIPER
877
0.7
E
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0.2
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50
IlXl
150
FRACTION NUMBER (Sml/tube)
Fla. 1. Gel filtration of Russell's viper venom on a Sephadex G-75 column (2.5 X 128 cm). Experimental conditions: sample volume, 5 ml per 100 mg of crude venom; elution buffer, 0.04 M Tris-HC1 buffer, pH 8.5, containing 0.1 M NaC1; flow rate, 50 ml/hr; fraction volume, 5 ml/tube. The fractions indicated by the solid bar were collected and lyophilized. Reproduced from H. Takahashi, S. Iwanaga, and T. Suzuki, I. Biochem. 76, 711 (1974) the adsorbed materials. Fractions of 5 ml are collected at 4 ° at a flow rate of 30 ml/hr. Figure 2 shows the elution pattern. Inhibitory activities were found in two peaks, and these fractions were combined and lyophilized (Fig. 2). Ammonium acetate is removed from the dried material in vacuo (0.005 mm Hg) at 40 °. The overall yields of RVV ~nhibitor I and I I are about 1 and 5 mg, respectively, from 100 mg of crude venom. The above procedures are reproducible and are applicable also for the isolation of proteinase inhibitors from the venoms of H e m a c h a t u s haemachatus 1° and N a j a nivea. ~ Properties 4,6 Purity. Both RVV inhibitor I and I I give a single band on disc polyacrylamide-gel electrophoresis in the presence and in the absence of sodium dodecyl sulfate (SDS). Physical' Properties. The molecular weights of both inhibitors I and I I are about 7200 (___1000), as estimated with SDS gel (7.5%) electro10H. Takahashi, S. Iwanaga, T. Kitagawa, Y. Hokama, and T. Suzuki, Proteinase Inhibitor, Proc. Int. Res. Con]., 2nd (Bayer Symp. V), Grosse Ledder, 1973, p. 265. 11T. Tatsuki, Y. Hokama, S. Iwanaga, and T. Suzuki, Seikagaku 46, 704 (1974). In Japanese.
878
NATURALLY OCCURRING PROTEASE INHIBITORS
1T ~.=.=.=
.=.= .=-=---x---=---N
---k---x---
x---
=.A-= "=
i
X---~---K---. .-'°
2
O.I
Io
J
0
0
~
200 FRACTION
300
[78]
u~ 20
I0
" I0
5
Z
I--. Z
_o
0
0
~
'
400
NUMBF'R f .~ml I h,h-t
FIG. 2. Purification of the Russell's viper venom proteinase inhibitor on a SESephadex column (2.0 × 30 cm). Experimental conditions: sample, the pooled fraction (15 mg) of Fig. 1; flow rate, 35 ml/hr; fraction volume, 5 ml/tube. A linear gradient elution starts with 500 ml each of 0.04 M ammonium formate buffer, pH 4.0, and 0.2 M ammonium acetate buffer, pH 9.5, and then a gradient of 0.2 M to 0.5 M buffer, pH 9.5, is used to elute all adsorbed material. Reproduced from H. Takahashi, S. Iwanaga, and T. Suzuki, F E B S Lett. 27, 207 (1972).
phoresis. The ultraviolet absorption spectra of these inhibitors are identical and show an absorption maximum at 278 nm and minimum at 256 nm at pH 9.0. The extinction coefficients of 1.0% polypeptide solutions of inhibitors I and II at 280 nm are 5.3 and 5.5, respectively. Stability. RVV inhibitors I and II are stable in the frozen and lyophilized state for at least one year and no loss of inhibitory activity is observed when inhibitor II is heated at pH 2 to 7.5 at 90 ° for 15 min. However, above pH 8.0 it is denatured. Specificity. The following enzymes are strongly inhibited by RVV inhibitors I and II: plasma and pancreatic kallikreins, trypsin, a-chymotrypsin, and plasmin from bovine source and human plasmin. The inhibitor II does not inhibit human and bovine thrombins, snake venom kininogenase, Reptilase, bromelain, ficin, papain, subtilisin BPN', thermolysin, and carboxypeptidases A and B. Kinetic Properties. RVV inhibitors I and II inactivate bovine trypsin, probably by formation an enzyme-inhibitor complex in a molar ratio of 1:1. The Ki values of inhibitor II, measured using synthetic ester substrate (TAME), are 0.76 nM for bovine trypsin, 0.14 nM (N ~acetyltyrosine ethyl ester) for bovine a-chymotrypsin; 0.29 nM for bovine plasma kallikrein, and 1.0 nM for bovine plasmin.
[78 !
879
PROTEINASE INHIBITORS FROM RUSSELL'S VIPER
TABLE I AMINO ACID COMPOSITIONSa OF PROTEINASE INHIBITORS ISOLATED FROM THE VENOMS OF RUSSELL'S VIPER, Hemachatus haemachatus, AND Naja nivea
Amino acid Lys His Ammonia Arg Asp Thr Ser Glu Pro Gly Ala Cys112 Val Met Ile Leu Tyr Phe Trp
Russell's viper, II
Russell's viper, I
3 2 (12) 7 8 3 2 5 2 8 2 6 1 0 1 3 3 4 0 60
2 2 (11) 5 7 2 2 5 5 7 3 4 1 0 2 1 1 3 0 52
H. haema- N. nivea chatus II II 2 1 (7) 5 5 3 1 6 2 6 5 6 1 0 3 4 3 4 0 57
2 2 (6) 7 4 3 1 6 2 6 5 6 1 0 3 3 2 4 -57
N. nwea Ia
N. nwea Ib
2 2 (7) 6 4 3 1 7 2 6 5 6 1 0 3 3 2 4 -57
4 1 (6) 4 5 3 2 4 3 3 4 6 3 1 3 3 2 3 -54
a The compositions were calculated (in residues per mole) by extrapolation or from the average values estimated on samples after 24-, 48-, and 72-hr hydrolyzates.
A m i n o Acid C o m p o s i t i o n 2 ,1° T h e c o m p o s i t i o n s of s e v e r a l i n h i b i t o r s i s o l a t e d f r o m t h e v e n o m s of R u s s e l l ' s v i p e r , H e m a c h a t u s haemachatus, a n d N a j a nivea a r e s h o w n in T a b l e I. C o m m o n f e a t u r e s i n c l u d e r e l a t i v e l y h i g h c y s t i n e c o n t e n t a n d f e w or no m e t h i o n i n e a n d t r y p t o p h a n r e s i d u e s ; no s u g a r r e s i d u e is d e t e c t e d in a n y i n h i b i t o r . A m i n o Acid Sequence. 7,s T h e s e q u e n c e s of R V V i n h i b i t o r I I a n d H H V i n h i b i t o r I I a r e s h o w n in F i g . 3, in c o m p a r i s o n w i t h t h o s e of b o v i n e p a n c r e a t i c t r y p s i n i n h i b i t o r 1~ a n d s n a i l i s o i n h i b i t o r K. 18 T h e o v e r a l l p r i m a r y s t r u c t u r e of s n a k e v e n o m p r o t e i n a s e i n h i b i t o r s is q u i t e s i m i l a r to t h o s e of t h e K u n i t z - t y p e i n h i b i t o r s , i n d i c a t i n g 5 0 - 6 0 % s e q u e n c e h o m o l o g i e s . M o r e o v e r , t h e 6 h a l f - c y s t i n e r e s i d u e s of t h e s e f o u r i n h i b i t o r s a r e in t h e s a m e p o s i t i o n s a l o n g t h e p o l y p e p t i d e s (Fig. 4). T h e r e a c t i v e sites of ~2B. Kassell, M. Radicevic, M. J. Anfield, and M. Laskowski, St., Biochem. Biophys. Res. Commun. 18, 255 (1965). ~3H. Tschesche, Angew. Chem. 86, 21 (1974).
880 RVV II HHV If SNAIL IK BPTI
NATURALLY OCCURRING PROTEASE INI-IIBITORS
[78]
I 0 15 ~ 20 Hls-Asp-Arg-Pco-TIP-Phe-Cys-Asn-Leu-Ala-Pro-G]u-Ser-G}y-Arg-Cys-Arg-G y-Hts-LeuArg-Pro-Asp-Phe-Cys-G]u-Leu-Pro-Ala-Glu-Thr-Gly-Leu-Cys-Lys-Ala-Tyr-liePyr-••y-A•g-P••-Asp-Phe-Cys-G]u-Leu-Pr•-A•a-••u-Th•-••y-P••-Cys-Lys-A•a-Ser-PheArg-Pro-Asp-Phe-Cys-Leu-G]u-Pro-Pro-Tyr-Thr-Giy-Pro-Cys-Lys-Ala-Arg-I]e-
5 30 5 40 Arg-Ar~-~e-Tyr~yr~Asn-Leu~B~u-Ser-Asn~Lys-~ys~Lys~a~-P~e-Phe-Tyr-G~y-G~y-CysArg-Ser-Phe-•is-Tyr-Asn-Leu-A•a-A•a-G•n-Lys-Cys-Leu-G]u•Phe-••e-Tyr-G•y-••y-CysAr~-G~n~Tyr-T~r-Tyr-Asn-Ser-Lys~Ser-~y-G~y-Cys-~n-G~n-Phe-~e-Tyr-G~y-~y-Cys~e-Arg-Tyr~Phe-Tyr-Asn-A~a-Lys-A~a-~y-Leu-Cys-~n-Thr-Phe-Va~-Tyr-G~y-~y-Cys45 50 5 G~y-G~y-Asn~A]a-Asn-Asn~Phe-G~u-Thr.~Arg-Asp-G~u-Cys-Ar~-G~u-Thr-Cys~G~y-G]y-L~ Giy-G]y-Asn-A]a-Asn-Arg-Phe-Lys-Thr-Ile-Asp-Glu-Cys-Arg-Arg-Thr-Cys-Va]-Gly Arg-Gly-Asn-Gln-Asn-Arg-phe-Asp-Thr-Thr-G]n-G]n-Cys-G]n-Gly-Va]-Cys-Yal
Arg-kla-Lys-Arg-Asn-Asn-Phe-Lys-Ser-Ala-Glu-Asp-Cys-Met-Arg-Thr-Cys-Gly-Gly-Ala FIG. 3. Comparison of the amino acid sequences of Russell's viper venom (RVV) inhibitor II, Hemachatus haemachatus (HHV) inhibitor II, snail isoinhibitor K, and bovine pancreatic trypsin inhibitor (Kunitz type). Based on H. Takahashi, S. Iwanaga, Y. tIokama, T. Suzuki, and T. Kitagawa, FEBS Left. 38, 217 (1974). B. Kassell, M. Radicevic, M. J. Anfield, and M. Laskowski, Sr., Bioehem. Biophys. Res. Commun. 18, 255 (1965) ; and H. Tschesche, Angew. Chem. 86, 21 (1974).
RVV inhibitor II and H H V inhibitor II are still unknown, but the most probable site for interaction with trypsin is the peptide linkage ArglTGlyl+ in the former, and Lysls-Ala~6 in the latter, because the homologies of all the inhibitors around these portions are extremely high. These results are very interesting in relation to the evolution of these inhibitors, since evolutionary histories of cows, snakes, and snails are quite different. I
i
FIG. 4. A schematic representation of the structure of the proteinase inhibitor isolated from the venom of Russell's viper. Based on T. Takahashi, S. Iwanaga, Y. Hokama, T. Suzuki, and T. Kitagawa, FEBS Let$. 38, 217 (1974) ; and H. Takahashi, S. Iwanaga, T. Kitagawa, Y. Hokama, and T. Suzuki, J. Biochem. 76, 721 (1974).
[79]
BROAD-SPECIFICITY INHIBITORS FROM SEA ANEMONES
881
TABLE II DISTRIBUTION OF PROTEINASE INHIBITORS IN VARIOUS SNAKE VENOMS a'b
Family
Bovine plasma kallikrein
Bovine plasma plasmin
Bovine pancreatic trypsin
Bovine pancreatic (~-chymotrypsin
12 40 88
18 91 110
5 22 74
5 77 217
100 100 100 4 125 215
62 12 42 20 34 43
55 3 60 22 18 28
80 15 20 13 34 105
Viperidae
V. russelli V. ammodytes B. arietans Elapidae
N. N. N. H. D. D.
hannah nivea haje haemachalus angusticeps polylepis
a From H. Takahashi, S. Iwanaga, a n d T. Suzuki, Toxicon 12, 193 (1974). b The values were expressed as the quantities (#g) to show 50 % inhibition of the activity of the proteinases. The following venoms showed no inhibitory activity when 100 t~g of each was used: V. pale~tinae, E. carinatu~, B. gabon@a, N. naja
atra, N. melanoleuca, N. nigricollzs, N. naja samarens~s, N. naja, B. fasczatus, B. mult~cinctus, L. semifasciata, L. lahcaudata, A. halys blomho~,, A. acutus, A. rhodostoma, A. p~scwo~us leucostoma, A. contortmx contortr~x, A. p~scivorus piscworus, A. contortr~x mokeson, C. atrox, C. adamanteus, C. viridis wr~dis, C. basiliscus, C. durissus terrificus, B. atrox, B. jararaca, T. flavoviridis, T. gramineus, T. okinavensis, T. mucrosquamalus, Causus rhombeatus.
Distribution2 ,14 Proteinase inhibitor of the same type as described here is demonstrated also in the venoms of several snakes of the Elapidae and Viperidac (Table II). However, it is not found in Crotalidae and Hydrophiidae venoms. ~4D. J. Strydom, Nature (London) New Biol. 243, 88 (1973).
[79] B r o a d - S p e c i f i c i t y I n h i b i t o r s f r o m Sea Anemones By GERT WUNDERER, L~SZLO BI~RESS, WERNER MACHLEIDT, and HANS FRITZ In the purification of neurotoxins from Anemonia sulcata, fractions with antitryptic activity were observed. 1 This inhibitory activity could be ascribed to a variety of basic polypeptides. ~,~ The broad specificity 1 L. B6ress and R. B~ress, Kiel. Meeres]orsch. 27, 117 (1971). : H. Fritz, B. Brey, and L. B6ress, Hoppe-Seyler's Z. Physiol. Chem. 353, 19 (1972). 3G. Wunderer, K. Kummer, H. Fritz, L. B~ress, and W. Machleidt, Proteinase Inhibitors, Proc. Int. Res. Con]., 2nd (Bayer Symp. V), Grosse Ledder, 1973, p. 277.
