Cellular oxidant stress and advanced glycation endproducts of albumin: caveats of the dichlorofluorescein assay.
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. These studies reveal important caveats of the widely used dichlorofluorescein assay and suggest methods other than the microtiter plate assay are needed to accurately assess cellular oxidant stress in presence of native or modified albumin.
Subramaniam
Ram
R
Institute of Pathology and Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Fan
Xing-Jun
XJ
Scivittaro
Vincenzo
V
Yang
Jianqi
J
Ha
Chung-Eun
CE
Petersen
Charles E
CE
Surewicz
Witold K
WK
Bhagavan
Nadhipuram V
NV
Weiss
Miriam F
MF
Monnier
Vincent M
VM
D000818:Animals; D001120:Arginine / Q000737:chemistry; D002118:Calcium / Q000378:metabolism; D002122:Calcium Chloride / Q000378:metabolism; D002460:Cell Line; D002645:Chickens; D002942:Circular Dichroism; D003300:Copper / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005452:Fluoresceins / Q000494:pharmacology*; D005456:Fluorescent Dyes / Q000494:pharmacology; D017127:Glycation End Products, Advanced / Q000737:chemistry* / Q000378:metabolism; D006868:Hydrolysis; D007501:Iron / Q000378:metabolism; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D018384:Oxidative Stress; D010100:Oxygen / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013997:Time Factors
D005452:Fluoresceins; D005456:Fluorescent Dyes; D017127:Glycation End Products, Advanced; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins; C081628:advanced glycation end products-bovine serum albumin; D012710:Serum Albumin, Bovine; C037631:2',7'-dichlorofluorescein; D003300:Copper; D001120:Arginine; D007501:Iron; D002122:Calcium Chloride; D010100:Oxygen; D002118:Calcium