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Staphylococcus aureus protease V8 (SPV8), also known as Endoproteinase Glu-C (EC 3.4.21.19), is an enzyme isolated from the bacteria Staphylococcus aureus. This unusual enzyme has been found to cleave specifically at glutamyl and aspartyl peptide bonds and has been used as a tool in the preparation of protein substrates for amino acid sequence analysis. SPV8 has been reported to show some stability toward various denaturants (Drapeau, G.R. (1977) Methods in Enzymology_, 47:189-191). In order to more adequately assess the denaturant stability of SPV8, the effect of guanidine hydrochloride (HC1), a common protein denaturant, on the proteolytic action of SPV8 was studied. The extent of cleavage of the glutamyl peptide bond in adrenocorticotropic hormone 1-10 (ACTH 1-10) was found to decrease with increasing concentrations of guanidine HC1.At 22°C in the presence of 3.0 M guanidine HCl, only 25% of SPV8's proteolytic activity was retained. In the presence of 4.0, 5.0, or 6.0 M guanidine HC1, virtually all proteolytic activity toward the glutamyl bond of ACTH 1-10 was lost, presumably due to the inactivation of the protease by denaturation or increased autolysis mediated by the guanidine HC1. At temperatures above 22°C, SPV8 was more susceptible to inactivation by guanidine HC1. Thus SPV8 appears to retain some proteolytic activity in the presence of guanidine HC1, but only at concentrations less than 4.0 M. There was no difference in the proteolytic activity of SPV8 toward the glutamyl peptide bond of ACTH 1-10 when incubation was carried out in ammonium bicarbonate buffer (pH 7.80), phosphate buffer (pH 7.80), or Tris-HC1 buffer (pH 7.80). The presence of 1 mM calcium chloride in the 3.0 M guanidine HC1/phosphate buffer solution enhanced the enzymatic action of SPV8. The presence of 1 mM calcium chloride in Tris-HC1 buffer (pH 7.80) does not effect the proteolytic activity of SPV8 at 22°C. However, there was slight reduction in SPV8's enzymatic action toward ACTH 1-10 when the 1 mM calcium chloride was present in the 3.0 M guanidine HC1/ammonium bicarbonate buffer (pH 7.80) solution. / Department of Chemistry