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 280nm 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.