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

High molecular weight serine-proteases have been identified in Loxosceles i ntermedia (brown spider) venom. The mechanism by which Loxosceles spp venom s 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 pre sent investigation, when we submitted L. intermedia venom to linear gradien t 3-20% SDS-PAGE stained by a monochromatic silver method we detected a het erogeneous protein profile in molecular weight, ranging from 850- to 5-kDa. In an attempt to detect zymogen molecules of proteolytic enzymes, venom al iquots were treated with several exogenous proteases. Among them, trypsin a ctivated two gelatinolytic molecules of 85- and 95-kDa in the venom. In exp eriments 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 functio n 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 o ptimal pH for their activities ranging from 7.0 to 8.0. Evaluation of the f unctional specificities of the 85- and 95-kDa venom proteases showed that t hese 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 involv ed in the harmful effects of the venom. (C) 2000 Elsevier Science Ltd. All rights reserved.