The precipitation and cross-linking of lens crystallins by ascorbic acid.
Bovine lens beta-crystallin was incubated with increasing concentrations of sugars and sugar derivatives for a period of 2 weeks in the dark at 37 degrees C. Marked protein precipitation and a browning reaction was observed with both ascorbic acid (ASA) and dehydroascorbic acid (DHA), but little or no reaction was seen with several other sugars and sugar analogs. Similar incubations were carried out with 20 mM ASA, 20 mM DHA and 20 mM glucose, but with increasing amounts of the individual crystallins. Glucose was capable of precipitating gamma-crystallin in the presence of air, but this reaction was decreased if dithiothreitol and a chelating agent were added prior to incubation. ASA and DHA produced precipitation and browning with gamma- and beta-crystallin, but not with alpha-crystallin or lens soluble proteins. Similar reactivities were observed both in air and under reducing conditions. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of these reaction mixtures showed little or no cross-linking with any of the lens proteins by glucose. ASA and DHA caused detectable dimer formation with gamma-crystallin, but produced the formation of dimers as well as highly polymerized proteins at the top of the gel with all the other crystallins and with lens soluble proteins. A time-course experiment with alpha-crystallin in the presence of air showed no cross-linking with 100 mM glucose over a 6-week period; however, 10 mM ASA caused definite cross-linking at 2 weeks, and at 6 weeks a dark smear of protein was visible throughout the gel. ASA was still capable of inducing cross-linking under low oxygen conditions but the protein smearing was markedly diminished. Further, the cross-linking pattern was similar to that seen in the water-insoluble fraction from older human lenses and cataracts. This reaction may be significant in vivo because cross-linking was observed under low-oxygen conditions with as little as 2 mM ASA, which is the level of ASA normally present in human lenses.
Ortwerth
B J
BJ
Mason Institute of Ophthalmology, Columbia, MO.
Feather
M S
MS
Olesen
P R
PR
D000818:Animals; D001205:Ascorbic Acid; D002417:Cattle; D011232:Chemical Precipitation; D003432:Cross-Linking Reagents; D003459:Crystallins; D003683:Dehydroascorbic Acid; D005947:Glucose; D013997:Time Factors
D003432:Cross-Linking Reagents; D003459:Crystallins; D005947:Glucose; D001205:Ascorbic Acid; D003683:Dehydroascorbic Acid