BetaA3/A1 crystallin from human cataractous lens contains an intramolecular disulfide bond

Abstract

PURPOSE. To develop an experimental approach that can identify amino acid sequences containing cysteine residues involved in disulfide bonding during cataractogenesis of the human lens. METHODS. Total proteins from cataractous and normal human lenses were solubilized anaerobically, followed by carboxymethylation of free sulfhydryl groups with iodoacetate. Carboxymethylated proteins were partially purified by reverse phase chromatography, then subjected to lys-C endoprotease digestion. Using reverse phase chromatography, each digest was resolved in the presence and absence of dithiothreitol (DTT), to identify peptides that were linked by disulfide bonds. These peptides were further characterized using a combination of Edman degradation and mass spectrometry. RESULTS. The reverse phase chromatography profiles of lys-C peptides from proteins of normal lenses were very similar in the presence and absence of dithiothreitol, while identical analysis of proteins from cataractous lenses demonstrated the presence of a lys-C peptide that corresponded to residues 163–193 of human betaA3/A1, with cysteine 170 and cysteine 185 linked via an intramolecular disulfide bond. CONCLUSIONS. Reverse phase chromatography of complex mixtures of lens protein digests, in the presence and absence of dithiothreitol, provides a rapid method of identifying sequences involved in disulfide bonding. The results of this analysis using the endoprotease lys-C have demonstrated that betaA3/A1 crystallin from cataractous lenses contains an inter-molecular disulfide bond involving cysteine residues 170 and 185.