Guanidine-stable chymoelastase : a comparative study of its hydrolytic specificity in the presence and absence of denaturant

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Jordan, Katherine Jo
Johnson, Eric R.
Issue Date
Thesis (M.S.)--Ball State University, 1987.
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Guanidine-stable chymoelastase (GUSCE), a component of the protease mixture known as Pronase, has been shown to be stable and active under conditions which denature and inactivate most proteins. In the absence of denaturant, this enzyme has shown proteolytic specificity for phenylalanyl, tyrosyl, and leucyl peptide bonds. In the presence of denaturant, however, the cleavage specificity has not been defined.In order to determine the effects of denaturant on the cleavage specificity of GUSCE, six small peptides of known amino acid sequence were hydrolyzed by GUSCE in the presence and absence of 6.OM guanidinium chloride. The site of GUSCE cleavage was acertained by dansylation of the new N-terminal amino acids, which were produced by proteolysis, followed by thin layer chromatographic identification of the resulting dansylated amino acids.The results indicate that GUSCE catalyzed the hydrolysis of phenylalanyl and tyrosyl peptide bonds in the absence as well as the presence of 6.OM guanidinium chloride. Of the tyrosyl and phenylalanyl peptide bonds hydrolyzed, all were between non-terminal amino acids, which illistrates the endo-peptidase characteristics of GUSCE. With one exception, only those peptide bonds cleaved by GUSCE in the absence of denaturant were cleaved in the presence of denaturant. In the case of oxytocin, the presence of denaturant was actually required for the cleavage of the Tyr(2)-Ile(3) peptide bond. The demonstrated predictablilty of GUSCE cleavage in the presence of denaturant should greatly enhance its utility in the sitespecific proteolysis of insoluble or otherwise proteolysisresistant protein substrates.Ball State UniversityMuncie, IN 47306