Cellular oxidant stress and advanced glycation endproducts of albumin: caveats of the dichlorofluorescein assay

Archives of Biochemistry and Biophysics
Ram SubramaniamV M Monnier

Abstract

In order to understand the mechanism by which advanced glycation endproducts (AGEs) elicit oxidative stress, macrophage-like RAW264.7 cells were exposed to various AGE-albumins, and oxidant stress was estimated from the fluorescence of oxidized dichlorofluorescein using the microtiter plate assay. Strongest fluorescence was observed with methylglyoxal modified albumin (MGO-BSA) compared with native albumin. Similar effects that were prevented by arginine coincubation were seen with phenylglyoxal-BSA. MGO-BSA had increased affinity for Cu(2+) and Ca(2+), but was conformationally similar to native albumin. Surprisingly, the mere addition of unmodified albumin to cells suppressed the fluorescence of oxidized DCF. While, several site-directed mutants of human serum albumin (HSA), including C34S and recombinant domains II and III retained fluorescence suppressing activity, proteolytic digests, recombinant domain I, and several nonalbumin proteins failed to suppress. Kinetic and ANS binding studies suggested albumin quenches DCF fluorescence by binding to hydrophobic pockets in domains II and III and that MGO-BSA is less hydrophobic than BSA. Finally, BSA also prevented H(2)O(2) catalyzed DCF fluorescence more potently than MGO-BSA. ...Continue Reading

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Jul 21, 2009·American Journal of Physiology. Heart and Circulatory Physiology·Lori S KangJudy M Muller-Delp
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