Quantification of the isomerization of Asp residue in recombinant human αA-crystallin by reversed-phase HPLC

• Published in: Journal of pharmaceutical and biomedical analysis Vol. 30; no. 6; pp. 1825 - 1833
• Main Authors: Sadakane, Yutaka, Yamazaki, Toshiaki, Nakagomi, Kazuya, Akizawa, Toshifumi, Fujii, Noriko, Tanimura, Takenori, Kaneda, Masaki, Hatanaka, Yasumaru
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 15.01.2003
• Subjects: alpha-Crystallin A Chain - analysis; Aspartic Acid - analysis; Chromatography, High Pressure Liquid - methods; Crystallin; Enantiomerization; Eye lens; Humans; Isoaspartate formation; Peptide Fragments - analysis; Recombinant protein; Recombinant Proteins - analysis; Stereoisomerism; Crystallin; Recombinant protein; Isoaspartate formation; Enantiomerization; Eye lens
• ISSN: 0731-7085; 1873-264X
• DOI: 10.1016/S0731-7085(02)00525-3

A method for determining the isomerization of Asp residues in proteins is described and demonstrated by quantifying the isomerization of Asp 151 in recombinant human αA-crystallin. First, four types of dodecapeptide fragment ( 146IQTGLD 151ATHAER 157) in which the Asp residue was either l-Asp, d-Asp, l-isoAsp or d-isoAsp were synthesized, and RP-HPLC conditions were established for their separation. Next, the Asp 151-containing peptide fragments isolated from the tryptic hydrolysate of recombinant αA-crystallin were analyzed under these conditions. New peaks, the retention times of which were the same as those of peptides containing d-Asp, l-isoAsp and d-isoAsp, were generated when αA-crystallin was incubated for 140 days at 37 °C. An amino acid composition, amino acid sequence, and enantiomeric analysis revealed that two peaks with retention times identical to those of peptides containing l-isoAsp and d-isoAsp represented dodecapeptide fragments containing l-isoAsp 151 and d-isoAsp 151, respectively. RP-HPLC analysis under other condition suggested that the peak with retention time identical to that of peptide containing d-Asp represented dodecapeptide fragments containing d-Asp 151. The present method does not require acid hydrolysis, which generates further isomerization products as artifacts, and thus make possible the sensitive quantification of each type of Asp isomer individually at a specific site in a protein. In our analysis of the Asp 151 residue in human αA-crystallin, the degree of isomerization from l-Asp to d-Asp can be determined to a level as low as 0.3%.