Lipid-protein modifications during ascorbate-Fe super(2+) peroxidation of photoreceptor membranes: protective effect of melatonin

• Published in: Journal of pineal research Vol. 41; no. 3; pp. 201 - 210
• Main Authors: Guajardo, Margarita H, Terrasa, Ana M, Catala, Angel
• Format: Journal Article
• Language: English
• Published: 01.10.2006
• ISSN: 0742-3098; 1600-079X
• DOI: 10.1111/j.1600-079X.2006.00352.x

The rod outer segment (ROSg) membranes are essentially lipoprotein complexes. Rhodopsin, the major integral protein of ROSg, is surrounded by phospholipids highly enriched in docosahexaenoic acid (22:6 n3). This fluid environment plays an important role for conformational changes after photo-activation. Thus, ROSg membranes are highly susceptible to oxidative damage. Melatonin synthesized in the pineal gland, retina and other tissues is a free radical scavenger. The principal aim of this work was to study the changes in the ROSg membranes isolated from bovine retina submitted to nonenzymatic lipid peroxidation (ascorbate-Fe super(2+) induced), during different time intervals (0-180 min). Oxidative stress was monitored by increase in the chemiluminescence and fatty acid alterations. In addition we studied the in vitro protective effect of 5 mm melatonin. The total cpm originated from light emission (chemiluminescence) was found to be lower in those membranes incubated in the presence of melatonin. The docosahexaenoic acid content decreased considerably when the membranes were exposed to oxidative damage. This reduction was from 35.5 plus or minus 2.9% in the native membranes to 12.65 plus or minus 1.86% in those peroxidized during 180 min. In the presence of 5 mm melatonin we observed a content preservation of 22:6 n3 (23.85 plus or minus 2.77%) at the same time of peroxidation. Simultaneously the alterations of membrane proteins under oxidative stress were studied using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Loss of protein sulfhydryl groups and increased incorporation of carbonyl groups were utilized as biomarkers of protein oxidation. In membranes exposed to Fe super(2+)-ascorbate, we observed a decrease of protein thiols from 50.9 plus or minus 3.38 in native membranes to 1.72 plus or minus 2.81 nmol/mg of protein after 180 min of lipid peroxidation associated with increased incorporation of carbonyl groups into proteins from 7.20 plus or minus 2.50 to 12.50 plus or minus 1.12 nmol/mg of protein. In the SDS-PAGE we observed a decrease in the content of all the proteins, mainly rhodopsin, as a consequence of peroxidation. Melatonin, prevent both lipid peroxidation and protein oxidation.