P 223 The glycation of bovine lens βL-, βS- and γ-crystallins demonstrated by frozen-sectioning and isoelectric focusing

Purtx~se: We like to demonstrate glycation of S,-, R,and y-crystallins in the microsectioned bovine lens, by isoelectric focusing using three staining methods. Methods: Pour bovine lenses of 1.185+0.131 years were frozen-sectioned into equa'cor and 11 layers. Watersoluble crvstallins were seuarated bv isoelectric focusing an8 stained: 1. with ?oomassie brilliant Blue (CBB); 2. with the lectin (LEc) Concanavalin-A, followed by horse-radish peroxidase (RRP) and diaminobenzidine (DAB): 3. for ulvcourotein receotors (REc). HRP and DAB. fiesuiis: crys&iiiis are separated i&o:'&, (I~-, R.-, a,-, 0, and y-crystallins. 1n thp lectin staining only BL-, R,and y-crystallins were positive, whereas the a&and I&-crystallins were? negative. Contrary to the glycated y-crystallins in the lens nucleus, the 13,and S,-crystallins were only glycated in the anterior cortex and to a somewhat lower extent also in the posterior cortical regions. Receptor staining was about 3 times less sensitive than the LEC! staining, but results were comparable. The degree of glycation: G, = LEC + CBB for y,-crystallins is mean 2.40,. for B,-crystallins 0.77 and for B,-crystallins 0.28. Conclusions: The degree of glycation of y-crystallins in the nucleus is 11 timeIs higher than cortical I?.,crystallins and 3.5 times higher than the cortical 13,crystallins. Application of Carver's model of chaperone activity gives the right interpretation of this study, because only glycated crystallins may fit into the central cavity (0 55A).