Abstract
Previous reports (Drescher, D.G., and Lee, K.S. (1978) Anal. Biochem. 84, 559-569; Lee, K.S., and Drescher, D.G. (1978) Int. J. Biochem. 9, 457-467) have shown that high performance liquid chromatographic analysis of amino acids with the o-phthaldialdehyde/2-mercaptoethanol reagent (OPA/2-ME) is one of the most sensitive procedures currently available for micro amino acid analysis. In the present paper, methods are presented for the modification of cysteine and cystine in proteins for micro amino acid analysis using OPA/2-ME. Cysteine and cystine, which both show low fluorescence with OPA/2-ME, are converted to cysteic acid with performic acid directly, or to S-3-sulfopropylcysteine with 1,3-propane sultone after reduction of cystine with tri-n-butylphosphine. Cysteic acid and S-3-sulfopropylcysteine form highly fluorescent adducts with OPA/2-ME. The formation of S-3-sulfopropylcysteine in proteins and the subsequent hydrolysis of the proteins with methanesulfonic acid are particularly useful for complete amino acid analysis at the picomole level using a single sample.
Highlights
Is one of the most sensitive procedures currently available for micro amino acid analysis
OPA/B-ME does not form fluorescent adducts with proline and forms only weakly fluorescent adducts with lysine, proline is accurately detected after oxidation with chloramine-T (5, lo), and lysine is detected as an adduct of intermediate fluorescence after adding the detergent Brij to the reaction mixture [2, 11]
For proteins treated with performic acid, cysteine and cystine were accurately determined because of the relatively high fluorescence of the cysteic acid-OPA/2-ME
Summary
Is one of the most sensitive procedures currently available for micro amino acid analysis. OPA/B-ME does not form fluorescent adducts with proline and forms only weakly fluorescent adducts with lysine, proline is accurately detected after oxidation with chloramine-T (5, lo), and lysine is detected as an adduct of intermediate fluorescence after adding the detergent Brij to the reaction mixture [2, 11]. Cysteine and cystine both form adducts of low fluorescence with the OPA reagent,’ but no in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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