tion of tyrosines in the presence of halides (3,4). More-Quantification of cysteines by amino acid composiover, this method requires several steps, including pertion analysis is inaccurate because of decomposition formic acid preparation immediately prior to oxidation, of these residues during protein hydrolysis. Cysteine lyophilization of the protein solution, oxidation of the (and cystine) residues are oxidized to cysteic acid folprotein lyophilizate with performic acid, removal of exlowing hydrochloric acid hydrolysis in the presence of cess performic acid usually by dilution and lyophilizasodium azide. Using selected native and recombinant tion, and finally hydrolysis with hydrochloric acid. If proteins, containing different numbers of cysteine resdetermination of residues modified during the oxidaidues, we investigated the conditions for the quantitation is desired, analysis must be performed twice, with tive oxidation of cysteines to cysteic acid in the presand without oxidation.
ence of sodium azide. Protein hydrolysis with
We recently reported the oxidation of cysteine and hydrochloric acid in the presence of 0.20% sodium cystine residues to cysteic acid following hydrochloric azide resulted in 87-100% oxidation of the cysteines acid hydrolysis in the presence of sodium azide (5). to cysteic acid which was easily quantified. The results Since this approach is very simple, only requiring the were highly reproducible so that the azide-induced oxpresence of sodium azide in the protein solution, we idation can be used as a general method to determine investigated whether the sodium azide-dependent oxicysteine residues in a given protein. The sodium azidedation could be used for quantitative detection of cysdependent oxidation is superior to oxidation with perteines. Here we report the determination of cysteine formic acid because (i) it can be performed in solution residues following protein hydrolysis with hydrochloric not requiring protein lyophilization and in approxiacid in the presence of sodium azide.
mately half of the time; (ii) it delivers slightly higher yields of cysteic acid; and (iii) it does not affect tyrosine residues, which can be modified during the per-MATERIALS AND METHODS formic acid treatment.