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Hydrolysis of peptides by Crotalidae and Viperidae venoms
Toxtcoe, 1968, Vol . S. pp. 201-205. PerQamon Press Ltd., Printed in Grcat $ritsin
HYDROLYSIS OF PEPTIDES BY CROTALIDAE AND VIPERIDAE VENOMS ANTHONY T . Ttr* and PAUL M . TOOM * Department of Chemistry, Utah State University, Logan, Utah, U.S .A . (Acceptedforpublication 1 May 1967)
Abstract-Peptidase activities of nine Viperidae and thirteen Crotalidae venoms were investigated using twenty-nine dipeptides, six tripeptides, and one tetrapeptide. Only five peptides were not hydrolyzed by any of the snake venoms investigated. Relatively few differences in substrate specificities were found for snake venoms regardless of species and geographical distribution . INTRODUCTION
IN CONTRAST to venom proteinases, peptidase activities of snake venoms have not been well characterized. Recent studies [l, 3] have indicated that many di- and tripeptides are hydrolyzed by various snake venoms. In order for further characterization of venom peptidases, venoms of Viperidae and Crotalidae not previously studied were investigated and the findings are reported in this communication . MATERIALS AND METHODS of Peptides were products Mann Research and Nutritional Biochemica.l s Corporation . Peptidase activities were detected by the paper chromatographic technique described previously [2-4]. Five mg peptide and 3 mg venom in 0~3 ml of 0~02M phosphate buffer at pH 7~0 was spotted on Whatman No. 1 filter paper and developed with butanol-acetic acid-water (4 :1 :5) and/or phenol-ammonia-water (80 :1 :19) . Amino acids and peptide spots were detected by spraying the papers with a 0~5 per cent solution ninhydrin in acetone. RESULTS AND DISCUSSION Of the twenty-nine dipeptides investigated only GIy-L-Asp, GIy-L-Glu, L-Leu-L-VaI, and L-Lys-Gly were not hydrolyzed by any of the snake venoms . With the exception of L-Leu-IrTyr and L-Leu-L-Phe, which were hydrolyzed by only some of the venoms, all other dipeptidea were hydrolyzed by all the venoms investigated . The difference in hydrolysis of L-Leu-r.-Tyr by the venoms of Agkistrodon acutus of Formosan origin and A. halys of Japanese origin is illustrated in Fig. 1. As may be seen from Table 1, the tripeptides were hydrolyzed by most of the snake venoms at both peptide linkages with only a few exceptions . Only one tetrapeptide (tetraglycine) was investigated and no hydrolysis was detected . In general, only a few differences in substrate specificities, regardless of species of snakes and geographical origins, could be found. 'Present address: Department of Biochemistry, Colorado State University, Fort Colline, Colorado, U.S.A . 201
202
ANTHONY T. TU and PAUL M. TOOM
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Hydrolysis of Peptides by Crotalidae and Viperidae Venons
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204
ANTHONY T. TU and PAUL M TOOM
O Z
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FIG. l . I"IYDROLYSIS OF LEU-L-L-TYR BY THE VENOM3 OF Agkistrodon acutus t.Nn A. ffalys. The original peptide, leucine, and tyrosine arerun as control. Venoms alone were also spotted as control and no ninhydrin positive spots were observed . Note the difference in the amino acid spots produced by the two venoms . Distinct spots of leucine and phenylalanine were produced after incubating the peptide with A. acutus venom. No such spots were observed for peptide incubated with A. hales venom. 1 . L-LeU-L-Tyr ; 2 and 3. A. acutus venom + L-Leu-L-Tyr ; 4 and 5. A. halys venom + L-Leu-L-Tyr ; 6. A. acutus venom ; 7. L-Leucine; 8. L-Tyrosine .
Toz. f.p . 204
Hydrolysis of Peptides by Crotalidae and Viperidae Venoms
205
Acknowledgements-This work was supported by NIH grant 1-ROl GM 14174. We wish to thank Mr . Josh M. MONROY-N~ro and Dr. J. M. Jn~xez-PoRxas for their generous supply of snake venoms . The technical assistance of Mr . WILLIAM H~xxts is appreciated . REFERENCES [1] Tu, A. T., CxuA, A, and JAMFS, G. P., Peptidase activities of snake venoms. Comp. Biochem . Pltysiol. 15, 517, 1965 . [2] Tu, A, T., Toots, P. M. and MuanocK, D. S., Chemical differences in the venorns of genetically different snakes . In : Anin~l Toxins (Ed. by RIJS.4ELL, F. E. and SAUxnsas, P. R.). Oxford : Pergamoa Press, p. 351, 1%7. [3] Tu, A. T. and Toots, P. M., Hydrolysis of peptides by snake venoms of Australia andNew Guinea, Aust.1. exp. Biol. »red. Sci. In press, 1968. [4] Tu, A. T. and MUItnocK, D. S., Protein nature and some enzymatic properties of lizard Heloderrrw snspectrun suspectum (Gila Monster) venom, Comp . Blochem. Physiol. 22, 389, 1 %7.
HYDROLYSIS OF PEPTIDES BY CROTALIDAE AND VIPERIDAE VENOMS ANTHONY T . Ttr* and PAUL M . TOOM * Department of Chemistry, Utah State University, Logan, Utah, U.S .A . (Acceptedforpublication 1 May 1967)
Abstract-Peptidase activities of nine Viperidae and thirteen Crotalidae venoms were investigated using twenty-nine dipeptides, six tripeptides, and one tetrapeptide. Only five peptides were not hydrolyzed by any of the snake venoms investigated. Relatively few differences in substrate specificities were found for snake venoms regardless of species and geographical distribution . INTRODUCTION
IN CONTRAST to venom proteinases, peptidase activities of snake venoms have not been well characterized. Recent studies [l, 3] have indicated that many di- and tripeptides are hydrolyzed by various snake venoms. In order for further characterization of venom peptidases, venoms of Viperidae and Crotalidae not previously studied were investigated and the findings are reported in this communication . MATERIALS AND METHODS of Peptides were products Mann Research and Nutritional Biochemica.l s Corporation . Peptidase activities were detected by the paper chromatographic technique described previously [2-4]. Five mg peptide and 3 mg venom in 0~3 ml of 0~02M phosphate buffer at pH 7~0 was spotted on Whatman No. 1 filter paper and developed with butanol-acetic acid-water (4 :1 :5) and/or phenol-ammonia-water (80 :1 :19) . Amino acids and peptide spots were detected by spraying the papers with a 0~5 per cent solution ninhydrin in acetone. RESULTS AND DISCUSSION Of the twenty-nine dipeptides investigated only GIy-L-Asp, GIy-L-Glu, L-Leu-L-VaI, and L-Lys-Gly were not hydrolyzed by any of the snake venoms . With the exception of L-Leu-IrTyr and L-Leu-L-Phe, which were hydrolyzed by only some of the venoms, all other dipeptidea were hydrolyzed by all the venoms investigated . The difference in hydrolysis of L-Leu-r.-Tyr by the venoms of Agkistrodon acutus of Formosan origin and A. halys of Japanese origin is illustrated in Fig. 1. As may be seen from Table 1, the tripeptides were hydrolyzed by most of the snake venoms at both peptide linkages with only a few exceptions . Only one tetrapeptide (tetraglycine) was investigated and no hydrolysis was detected . In general, only a few differences in substrate specificities, regardless of species of snakes and geographical origins, could be found. 'Present address: Department of Biochemistry, Colorado State University, Fort Colline, Colorado, U.S.A . 201
202
ANTHONY T. TU and PAUL M. TOOM
-I- ~-+~~ + -I ++ +-~+ ~ r
-f- -F-I -+ . .t .~.-i .E ._~
~- -I- + -I--F+~- ++++ +-I-
-F ++ -F + -~ + +-I-
s s + +~ --I- ~F +++++ +-F+
+ +~ --I-+++ +-i-
Y ~ ~ y 9 `~ 9 3 ß B k ~ ++++++++++++
++++i-++++
+-I-+++++++++++
+-++++++++
IIIIIIIIII111
IIIIIIIII
vd
-I- ++++ -F +++++++
+++++++++
;, a
IIIIIIIIIIIII
IIIIIIIII
++++ -I- +++ +-~ +++
+-F-+++++++
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9
6
$
8
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ô m
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a a~ _T
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y y 1~ ,..
.J ~ â ci ci d .. ~ ".~ ~o,`~~~C`C`~C~~41~i U
ô OS FiFiE~i
9
Q
o
~1 , ~~~~ V
Hydrolysis of Peptides by Crotalidae and Viperidae Venons
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+++++++++++++ s s s s s 9 +++++++++++++
+ -i-+~-+++++ .
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203
204
ANTHONY T. TU and PAUL M TOOM
O Z
IIIIIIIIIIIII
IIIIIIIII
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FIG. l . I"IYDROLYSIS OF LEU-L-L-TYR BY THE VENOM3 OF Agkistrodon acutus t.Nn A. ffalys. The original peptide, leucine, and tyrosine arerun as control. Venoms alone were also spotted as control and no ninhydrin positive spots were observed . Note the difference in the amino acid spots produced by the two venoms . Distinct spots of leucine and phenylalanine were produced after incubating the peptide with A. acutus venom. No such spots were observed for peptide incubated with A. hales venom. 1 . L-LeU-L-Tyr ; 2 and 3. A. acutus venom + L-Leu-L-Tyr ; 4 and 5. A. halys venom + L-Leu-L-Tyr ; 6. A. acutus venom ; 7. L-Leucine; 8. L-Tyrosine .
Toz. f.p . 204
Hydrolysis of Peptides by Crotalidae and Viperidae Venoms
205
Acknowledgements-This work was supported by NIH grant 1-ROl GM 14174. We wish to thank Mr . Josh M. MONROY-N~ro and Dr. J. M. Jn~xez-PoRxas for their generous supply of snake venoms . The technical assistance of Mr . WILLIAM H~xxts is appreciated . REFERENCES [1] Tu, A. T., CxuA, A, and JAMFS, G. P., Peptidase activities of snake venoms. Comp. Biochem . Pltysiol. 15, 517, 1965 . [2] Tu, A, T., Toots, P. M. and MuanocK, D. S., Chemical differences in the venorns of genetically different snakes . In : Anin~l Toxins (Ed. by RIJS.4ELL, F. E. and SAUxnsas, P. R.). Oxford : Pergamoa Press, p. 351, 1%7. [3] Tu, A. T. and Toots, P. M., Hydrolysis of peptides by snake venoms of Australia andNew Guinea, Aust.1. exp. Biol. »red. Sci. In press, 1968. [4] Tu, A. T. and MUItnocK, D. S., Protein nature and some enzymatic properties of lizard Heloderrrw snspectrun suspectum (Gila Monster) venom, Comp . Blochem. Physiol. 22, 389, 1 %7.