Identification of high molecular weight serine-proteases in Loxosceles intermedia (brown spider) venom

High molecular weight serine-proteases have been identified in Loxosceles intermedia (brown spider) venom. The mechanism by which Loxosceles spp venoms cause dermonecrotic injury (a hallmark of loxoscelism) is currently under investigation, but it seems to be molecularly complex and in some instance...

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Bibliographic Details
Published in:	Toxicon (Oxford) Vol. 38; no. 6; pp. 825 - 839
Main Authors:	Veiga, Silvio S., da Silveira, Rafael B., Dreyfuss, Juliana L., Haoach, Juliana, Pereira, Aline M., Mangili, Oldemir C., Gremski, Waldemiro
Format:	Journal Article
Language:	English
Published:	Oxford Elsevier Ltd 01-06-2000 Elsevier Science
Subjects:	Animal poisons toxicology. Antivenoms Animals Biological and medical sciences Electrophoresis, Polyacrylamide Gel - methods enzyme activity Female gelatin Gelatin - metabolism Hydrogen-Ion Concentration Hydrolysis Loxosceles Loxosceles intermedia Medical sciences Mice Mice, Inbred C57BL Molecular Weight Phosphoric Diester Hydrolases - chemistry Phosphoric Diester Hydrolases - toxicity polyacrylamide gel electrophoresis protease proteinase inhibitors proteinases proteolysis proteolytic activation Rabbits Serine Endopeptidases - chemistry Serine Endopeptidases - toxicity serine proteinase Serine Proteinase Inhibitors - pharmacology serine proteinases Spider Venoms - chemistry Spider Venoms - enzymology Spider Venoms - toxicity Substrate Specificity Toxicology Trypsin - pharmacology venoms Enzyme Toxicity Animal origin Identification Protein Peptidases Arachnida Proteolysis Arthropoda Venom Hydrolases Skin Invertebrata Mechanism of action
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Summary:	High molecular weight serine-proteases have been identified in Loxosceles intermedia (brown spider) venom. The mechanism by which Loxosceles spp venoms cause dermonecrotic injury (a hallmark of loxoscelism) is currently under investigation, but it seems to be molecularly complex and in some instance proteases might be expected to play a role in this skin lesion. In the present investigation, when we submitted L. intermedia venom to linear gradient 3–20% SDS-PAGE stained by a monochromatic silver method we detected a heterogeneous protein profile in molecular weight, ranging from 850- to 5-kDa. In an attempt to detect zymogen molecules of proteolytic enzymes, venom aliquots were treated with several exogenous proteases. Among them, trypsin activated two gelatinolytic molecules of 85- and 95-kDa in the venom. In experiments of hydrolysis inactivation using different protease inhibitors for four major class of proteases, we detected that only serine-type protease inhibitors were able to inactivate the 85- and 95-kDa enzymes in the venom. An examination of the 85- and 95-kDa gelatinolytic activities as a function of pH showed that these proteases had no apparent activities at pH below 5.0 and higher than 9.0 and displayed little activity at pH 6.0, with the optimal pH for their activities ranging from 7.0 to 8.0. Evaluation of the functional specificities of the 85- and 95-kDa venom proteases showed that these proteases efficiently degrade gelatin (denatured collagen) but have no proteolytic activity on hemoglobin, immunoglobulin, albumin, fibrinogen or laminin, suggesting specificity of their proteolytic actions. We describe here two serine-proteases activities in L. intermedia venom probably involved in the harmful effects of the venom.
Bibliography:	ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23
ISSN:	0041-0101 1879-3150
DOI:	10.1016/S0041-0101(99)00197-X