GLYCATION OF HUMAN LENS PROTEINS - PREFERENTIAL GLYCATION OF ALPHA-A SUBUNITS

Glycation of crystallins and high molecular weight (HMW) aggregates was followed during aging (16-85 years) and in diabetes (44 and 70 years old). Lens soluble and insoluble fractions were reduced with [3H]NaBH4 and separated by molecular sieve HPLC. The protein content in each HPLC peak was measured by the Lowry method. The tritium incorporation, expressed as cpm mg-1 protein, was taken as a measure of early glycation and specific non-tryptophan fluorescence (Ex: 370 nm; Em: 440 nm), expressed as relative fluorescence U mg-1 protein, was taken as a measure of advanced glycation. The youngest lenses analysed were 16 and 17 years old and these provided the baseline values. The results showed that during aging there was about a three-fold increase in tritium incorporation and fluorescence of α-crystallin, while the increases in β and γ were only two-fold from the levels seen in 16- and 17-year-old lenses. On the other hand, both the soluble and insoluble HMW aggregate fractions showed up to five-fold increase in tritium incorporation during aging. The fluorescence was about two-fold higher in the insoluble HMW aggregates as compared to the soluble HMW aggregates in 16- and 17-year-old lenses and both showed an increase of about three-fold during aging. Diabetes resulted in an approximately 10-50% increase in tritium incorporation and non-tryptophan fluorescence of various crystallins and HMW aggregates. Since α-crystallins showed a higher level of glycation than other crystallins, and soluble HMW aggregates appear to have consisted predominantly of α-crystallins, we further determined the level of glycation of α-crystallin subunits. Alpha-crystallins from 50-85-year-old lenses was used to separate the subunits by both reverse-phase HPLC (RP-HPLC) and gel electrophoresis. Determination of the protein bound radioactivity showed that αA was more glycated than αB. Furthermore, the RP-HPLC also separated a modified α peak, in addition to αA and αB, which presumably originated from HMW aggregates. The modified α had an even higher level of early glycation than αA. Interestingly, the fluorescence associated with the modified α was also several fold higher than αA and αB. © 1992.