Uses
A protease that cleaves proteins on the carboxyl bond of arginine
Uses
Clostripain from
Clostridium histolyticum has been used as a proteolytic enzyme in perfusate to detect its effect on tube hematocrit. It has also been used in limited proteolysis of DNA polymerase (gp43) of phage T4 (RB69 gp43).
Biochem/physiol Actions
Clostripain from Clostridium histolyticum is composed of two polypeptide chains, with molecular masses of 41.7 kDa and 15.4 kDa. Clostripain has a highly restricted substrate specificity for Arg-Xaa peptide bonds. Therefore, clostripain has been explored as a potential enzyme for protein sequencing purposes. It has also been studied as a catalyst for condensation of pharmaceutically important peptides containing Arg-Pro bonds.
Purification Methods
Clostripain is isolated from Clostridium histolyticum callogenase by extraction in pH 6.7 buffer, followed by hydroxylapatite chromatography with a 0.1-0.2 M phosphate gradient, then Sephadex G-75 gel filtration with 0.05M phosphate pH 6.7, dialysis and a second hydroxylapatite chromatography (gradient elution with 0.1M 0.3M phosphate, pH 6.7). It has proteinase and esterase activity and is assayed by hydrolysing N-benzoyl-L-arginine methyl ester. [Mitchell & Harrington J Biol Chem 243 4683 1968, Methods Enzymol 19 635 1970.]
