Clostripain-catalyzed re-formation of a peptide bond in a cytochrome C fragment complex.
Enzymatically-catalyzed condensation of cytochrome c fragments, ferrous heme fragment (1-38) and apofragment (39-104), has allowed the back-conversion of cytochrome c complex to native cytochrome c. The conversion was accomplished in 90% (v/v) glycerol, a solvent which has been shown to decrease the ionization of the terminal alpha-carboxyl group liberated during hydrolysis of a peptide bond. The effect on the pK is probably the main reason the thermodynamic obstacle to re-synthesis is minimized. A 30% conversion to cytochrome c was obtained. The cytochrome c product was distinguished from the non-covalent complex and separated fragments by molecular weight analysis with sodium dodecyl sulfate polyacrylamide gel electrophoresis, by elution from Sephadex G-50 and sulfopropyl-Sephadex in the presence of denaturant, by amino acid analysis of the product purified under complex-dissociation conditions, and by spectral analysis of the absorption bands of the heme. This method provides an opportunity to study the covalent rather than the complex form of cytochrome c analogs.
D000596:Amino Acids / Q000032:analysis; D002850:Chromatography, Gel; D003546:Cysteine Endopeptidases; D003574:Cytochrome c Group / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism*
D000596:Amino Acids; D003574:Cytochrome c Group; D010446:Peptide Fragments; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; C019913:clostripain