Journal of chromatography. B, Analytical technologies in the biomedical and life sciences

Determination of rate constants for β-linkage isomerization of three specific aspartyl residues in recombinant human αA-crystallin protein by reversed-phase HPLC.

PMID 21470922


The major soluble eye lens protein, αA-crystallin, has a very long half-life. Thus, many post-translational modifications, including stereoinversion, have been found in its constituent amino acids. We determine the rates of β-linkage isomerization, which is the main reaction through the formation of a succinimide intermediate, of specific Asp residues of recombinant human αA-crystallin protein by simple RP-HPLC method. Kinetic analyses of the β-linkage isomerization were performed on the three Asp residues of αA-crystallin, (58)Asp, (84)Asp, and (151)Asp, because the d/l ratios of both the (58)Asp and (151)Asp residues were higher than 1.0 in the αA-crystallin isolated from aged human eye lens. The β-linkage isomerizations of both the (58)Asp and (84)Asp residues were suppressed in the recombinant protein by approximately 0.4-0.5 times compared to those in the synthetic peptide below 50 °C, whereas the isomerization of the (151)Asp residue occurred at the same rate for the whole protein and synthetic fragmentary peptide. The suppression of (58)Asp isomerization in the recombinant protein relaxed to some extent when the αA-crystallin protein was incubated at a high temperature. The far-UV CD spectra showed that the secondary structure of the protein was partially disordered at temperatures greater than 60 °C in the recombinant αA-crystallin protein. These results suggest that the (58)Asp residue was restrained from forming the succinimide intermediate by the higher order structure of the αA-crystallin protein, and that the structural environment around the (151)Asp residue of the αA-crystallin was similar to that of the synthetic fragmentary peptide with respect to succinimide formation. The difference in the influence of the secondary structure of the αA-crystallin protein inverts the order of the succinimide formations of the (58)Asp and (151)Asp residues in the recombinant protein as compared with the order in the synthetic fragmentary peptides.

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