Bromopyruvate as an Affinity Label for Baker's Yeast Flavocytochrome b2

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Abstract
Bromopyruvate behaves as an active-site-directed reagent for flavocytochrome b2 (Mulet and Lederer 1977) some unspecific labeling occurred. Radioactive peptides were purified after labeling the enzyme with bromo[2-14C]pyruvate. Direct proteolysis of the labeled enzyme led to a multiplicity of labeled peptides, due to incomplete proteolysis. Four of them were characterized, corresponding to 2 unique cysteine residues. Cyanogen bromide cleavage of the labeled protein, followed by enzymatic digestion, led to the isolation of peptides corresponding to 4 cysteines, including the 2 previously identified ones. Apparently, the active-site cysteine must be the one present in the 85-residue cyanogen bromide peptide .alpha.CB3. The sequence around that cysteine is Ala-Ser-Cys-Ser-Pro-Gln-Gln-Ile-Ile-Glu-Ala-Ala-.