Reverse-phase HPLC analysis of human alpha crystallin

A rapid and highly sensitive reverse-phase HPLC (RP-HPLC) method was used to separate crystallin subunits from human alpha crystallin. Three distinct peaks were separated; by electrophoretic and immunological analyses the first and second peaks were identified as alpha B and alpha A respectively. On the other hand, peak 3 appeared to be a modified form of alpha crystallin. The ratio of alpha A and alpha B proteins was 3:1 in 1 day old lenses which gradually changed to 2:1 in 17 year old lenses and to 1:1 in the 50 and 82 year old whole lenses and 82 year old lens cortex, with a concomitant increase in the modified alpha, suggesting that alpha A subunits are relatively more involved in aggregation. Analysis of the 82 year old lens nucleus also supported this conclusion. The RP-HPLC analysis of the HMW aggregate fraction showed substantial enrichment of the modified alpha. The alpha A and alpha B subunits independently reassociated to form polymeric alpha crystallin whereas the modified alpha reassociated to form HMW aggregates as shown by molecular sieve HPLC. Hence it appears that the HMW aggregate peak was constituted by modified alpha crystallin. Only in the peak 3 material the 280 nm absorbance was about 2-fold higher than what was expected from the actual protein content. The data suggest that the changes induced by post-translational modifications may have some role in the formation of modified alpha. The present RP-HPLC method is useful in separating these modified alpha from the unmodified alpha A and alpha B subunits.