The glycation and cross-linking of isolated lens crystallins by ascorbic acid

Abstract

Listen

Translate article

Individual lens crystallins were isolated from calf lens extracts and incubated in the presence of ascorbic acid for 3 weeks under aerobic conditions. Both α-crystallin and β H-crystallin rapidly cross-linked to form high molecular weight proteins, which did not enter the resolving gel on SDS-PAGE. Beta l -crystallin was somewhat less reactive, but γ-crystallin showed little or no crosslinking. Gamma-crystallin, however, was almost equivalent to the other crystallins as a substrate for glycation. This was measured by: ( a) the binding of protein to a boronate affinity column; ( b) the incorporation of 3H from NaB 3H 4 into protein; ( c) amino acid analysis of the modified proteins to estimate the extent of lysine modification; and ( d) the incorporation of [1- 14C]ASA into individual crystallins. When the separated crystallins were combined with [ 125I]γ-crystallin and incubated with ascorbic acid, radioactivity was readily incorporated into the cross-linked products with other crystallins, but again not with γ-crystallin itself. Gel filtration chromatography of a mixture of [ 125I]γ-crystallin and α-crystallin showed the formation of a complex between γ- and α-crystallins. These data suggest that all crystallins are glycated, but that cross-linking occurs preferentially between proteins, which are already bound together non-covalently.