Correlation between the loss of the chaperone-like activity and the oxidation, isomerization and racemization of gamma-irradiated alpha-crystallin

Alpha-crystallin possesses a molecular chaperone-like activity that prevent s proteins from aggregating; however, the mechanism of this activity is not well known. Here we have taken gamma-irradiated alpha-crystallin and studi ed the relationship between the decrease in chaperone-like activity and the modifications such as oxidation, isomerization and racemization of amino a cids in this molecule. We found that the chaperone-like activity of alpha-c rystallin decreased with increasing gamma irradiation. After 4000 Gy gamma irradiation the activity of alpha-crystallin was reduced to 40% of the leve l of nonirradiated, native alpha-crystallin. The circular dichroism spectru m showed that the secondary structure of the irradiated alpha-crystallin ha d not changed. However, its tertiary structure appeared to change following more than 1000 Gy irradiation. Sodium dodecyl sulfate-polyacrylamide gel e lectrophoresis also indicated that cross-linking of alpha-crystallin increa sed with increasing radiation doses. Irradiated and nonirradiated alpha-cry stallin was subjected to trypsin digestion and peptide analysis by reverse- phase high-performance liquid chromatography and mass and sequence analysis . Depending on the radiation dose, Met-1 of alpha A-crystallin was oxidized to methionine sulfoxide. In addition, Asp-151 of alpha A-crystallin was is omerized to the beta-Asp form after irradiation, and racemization of Asp-15 1 decreased. Thus, the loss of the chaperone-like activity of alpha-crystal lin is related to changes in its isomerization, oxidation and racemization.