Cysteine and cystine: High-performance liquid chromatography of o-phthalaldehyde derivatives

Abstract

Publisher Summary This chapter discusses the High-Performance Liquid Chromatography (HPLC) of o-phthalaldehyde derivatives. o- Phthalaldehyde reacts rapidly with amino acids at normal ambient temperatures under aqueous conditions to yield intensely fluorescent derivatives. The relatively low polarity of these derivatives is ideally suited to separation by reversed-phase HPLC with a sensitivity of 1 pmol for each amino acid fluorophore. Because of the excess thiol present in the prepared o-phthalaldehyde reagent, the derivative of a sulfhydryl amino acid is the reduced amino acid isoindole. Thus, cystine or cysteine will yield the same isoindole derivative. The fluorescence intensity of cysteine isoindole can be as little as 2% of that of other amino acid isoindoles. Methods used to increase the fluorescence yield of the cysteine isoindole have included the oxidation of cysteine to cysteic acid with performic acid s and the reaction of the surfhydryl of cysteine with 4-vinylpyridine, ethyleneimine, or iodoacetate. Cysteine derivatives formed with these reagents subsequently yield isoindoles that have approximately the same fluorescence intensity as other amino acid isoindoles. However, the alkylation of cysteine with iodoacetate to form S-carboxymethylcysteine has the advantages of being a rapid reaction at ambient temperatures and compatible with the o-phthalaldehyde reaction. Other alkylating agents such as iodoacetamide can be used to alter the retention of the isoindole on the HPLC column if required.