143] Isolation and purification of flavin peptides

Abstract

Publisher Summary This chapter focuses on the isolation and purification of flavin peptides and their properties are reviewed. The flavin prosthetic group of mammalian succinate dehydrogenase is covalently bound to the protein. Only proteolytic digestion separates the flavin from the protein core and solubilizes a dinucleotide bound to peptides of various dimensions. Purified beef heart succinate dehydrogenase is digested with protcolytic enzymes. The soluble peptides produced are fractioned by solvent extraction and gel filtration until the peptide bound flavin is separated from all other nonflavin peptides. The steps involved in the preparation are: (1) proteolysis, (2) chromatography on sephadex, (3) extraction with p -cresol and hydrolysis, (4) chromatography on biogel, and (5) chromatography on silica gel. Flavin peptides are quantified spectrophotometrically or fluorometrically. The fluorometric determination is based on the fact that flavin peptides emit fluorescence only in weakly acidic medium. The purity of a preparation may be estimated by calculating the ratios of absorbancies at wavelengths corresponding to maxima and to minima in the pure compound and comparing them to the proper values of the pure flavin peptide. Paper electrophoresis is used to assay purity with respect to free flavins.