Circular Dichroism and Gel Filtration Behavior of Subtilisin Enzymes in Concentrated Solutions of Guanidine Hydrochloride

Michael F. Brown, Thomas Schleich

Research output: Contribution to journalArticle

12 Scopus citations

Abstract

The circular dichroism of diisopropylphosphorylsubtilisins Novo and Carlsberg in both the near- and farultraviolet spectral regions is unaltered by concentrations of guanidine hydrochloride as high as 4 M at neutral pH. At concentrations of guanidine hydrochloride greater than 4 M slow irreversible time-dependent changes, apparently obeying second-order kinetics, are evident in both the near- and far-ultraviolet circular dichroism of these enzymes. Gel filtration studies of inactivated subtilisin enzymes reveal the circular dichroism changes to be accompanied by the appearance of aggregated protein material. The changes in circular dichroism and the production of associated subtilisin species are sensitive to protein concentration, denaturant concentrations, and pH. The circular dichroism of active subtilisins Novo and Carlsberg in guanidine hydrochloride exhibits irreversible changes similar to those observed for the inactivated subtilisins. Aggregated protein material is also formed initially in the presence of guanidine hydrochloride, but is rapidly autolyzed to low molecular weight fragments.

Original languageEnglish (US)
Pages (from-to)3069-3074
Number of pages6
JournalBiochemistry
Volume14
Issue number14
DOIs
StatePublished - Jul 1 1975
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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