Derivatization of cysteine and cystine for fluorescence amino acid analysis with the o-phthaldialdehyde/2-mercaptoethanol reagent.
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.
D000447:Aldehydes; D000596:Amino Acids / Q000032:analysis*; D002256:Carbonic Anhydrases; D003545:Cysteine; D003553:Cystine; D006801:Humans; D007202:Indicators and Reagents; D008623:Mercaptoethanol; D009113:Muramidase; D013050:Spectrometry, Fluorescence / Q000379:methods; D009764:o-Phthalaldehyde
D000447:Aldehydes; D000596:Amino Acids; D007202:Indicators and Reagents; D003553:Cystine; D008623:Mercaptoethanol; D009764:o-Phthalaldehyde; D009113:Muramidase; D002256:Carbonic Anhydrases; D003545:Cysteine