Open AccessBinding of Cibacron Blue F3GA to Flavocytochrome b 2 from Baker's Yeast
Author(s) -
POMPON Denis,
LEDERER Florence
Publication year - 1978
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1978.tb12636.x
Subject(s) - flavin group , chemistry , cofactor , ionic strength , ethylene glycol , elution , flavin adenine dinucleotide , sephadex , enzyme , stereochemistry , chromatography , biochemistry , organic chemistry , aqueous solution
1 Flavin‐free cytochrome b 2 has been prepared by rapid Sephadex filtration at acid pH. The method, which yields an apo‐enzyme with high recontitution potential and has several advantages over preiviously used procedures, is described in detail. 2 Flavin‐free cytochrome b 2 thus prepared is retained by blue‐dextran‐bound Sepharose. It can be eluted by an increase in ionic strength, by dilute ethylene glycol and specifically by low concentrations of FMN. The holoenzyme is not retarded at all. 3 Both flavin‐free and holocytochrome b 2 bind Cibacron blue F3GA with appearance of distinct difference spectra. Cibacron blue is an inhibitor for the holoenzyme, it shows mixed type inhibition with respect to lactate. 4 It is concluded that there are two types of binding sites for Cibacron blue F3GA on flavocytochrome b 2 . Both possess ionic and hydrophobic character; one of them, which is the flavin binding site, is only available in the absence of the cofactor.Taken together these results may mean that the enzyme possesses a local flavin‐binding structure similar to the ‘dinucleotide fold’.