Simultaneous racemization and isomerization at specific aspartic acid residues in αB-crystallin from the aged human lens

Abstract

We provide evidence that the racemization and isomerization of aspartyl(Asp) residues occur simultaneously in the αB-crystallin in the lens of aged (mean age: 80 years) and young (age: 11 months) humans. We purified αB-crystallin and subjected it to tryptic digestion. The resulting peptides were separated by reverse-phase high-performance chromatography (RP-HPLC) and were characterized by amino-acid composition, sequence analysis and mass spectrometry. Two specific sites, Asp-36 ( d l of Asp: 0.92) and Asp-62( d l of Asp: 0.57), among 13 Asp/asparginyl (Asn) residues in aged αB-crystallin, were found to be highly racemized and isomerized to form β-Asp residues. The β-Asp-containing peptides were clearly distinguished from normal Asp-containing (α-Asp) peptides by RP-HPLC. The racemization and isomerization of Asp residues in aged αB-crystallin may occur via a succinimide intermediate. In young αB-crystallin, we observed neither racemization nor isomerization. We also found that Met-68 was oxidized to form Met sulfoxide to a greater extent in aged αB-crystallin than in young αB-crystallin. We concluded that racemization, isomerization, and oxidation of αB-crystallin occur spontaneously in the aging process.