Sinapinic acid can replace ascorbate in the biotin switch assay.
BACKGROUND
Protein S-nitrosation is an important post-translational modification altering protein function. Interaction of nitric oxide with thiols is an active area of research, and is one of the mechanisms by which NO exerts its biological effects. Biotin switch assay is the method, which has been developed to identify S-nitrosated proteins. The major concern with biotin switch assay includes reducing disulfide which may lead to false positives. We report a modification of the biotin switch assay where sinapinic acid is utilized instead of ascorbate to eliminate potential artifacts in the detection of S-nitrosated proteins.


METHODS
The denitrosation ability of sinapinic acid was assessed by monitoring either the NO or NO(2)(-) released by chemiluminescent NO detection or by the griess assay, respectively. DTNB assay was used to compare disulfide reduction by ascorbate and sinapinic acid. Sinapinic acid and ascorbate were compared in the biotin switch detection of S-nitrosoproteins in RAW 264.7 cells+/-S-nitrosocysteine (CysNO) exposure.


RESULTS
We show that sinapinic acid has the ability to denitrosate S-nitrosothiols at pH 7.0 and denitrate plus denitrosate at pHs 8 and 8.5. Unlike ascorbate, sinapinic acid degrades S-nitrosothiols, but it does not reduce disulfide bridges.


CONCLUSIONS
Sinapinic acid denitrosate RSNO and does not reduce disulfides. Thus can readily replace ascorbate in detection of S-nitrosated proteins in biotin switch assay.


CONCLUSIONS
The work described is important in view of protein S-nitrosation. In this study we provide an important modification that eliminates artifacts in widely used technique for detecting the S-nitrosoproteome, the biotin switch assay.
Kallakunta
Vasantha Madhuri
VM
Department of Chemistry and Biochemistry, 275-1, EH, University of Windsor, 401 Sunset Avenue, Windsor, Ontario N9B3P4, Canada.
Staruch
Andrea
A
Mutus
Bulent
B
D000818:Animals; D001205:Ascorbic Acid / Q000737:chemistry / Q000378:metabolism*; D001710:Biotin / Q000737:chemistry / Q000378:metabolism*; D015153:Blotting, Western; D002460:Cell Line; D003373:Coumaric Acids / Q000737:chemistry / Q000378:metabolism*; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D004228:Dithionitrobenzoic Acid / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008163:Luminescent Measurements / Q000379:methods; D008264:Macrophages / Q000166:cytology / Q000378:metabolism; D015394:Molecular Structure; D009569:Nitric Oxide / Q000737:chemistry / Q000378:metabolism*; D009573:Nitrites / Q000737:chemistry / Q000378:metabolism; D015538:Nitrosation; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D015203:Reproducibility of Results; D026403:S-Nitrosothiols / Q000737:chemistry / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism
D003373:Coumaric Acids; D004220:Disulfides; D009573:Nitrites; D011506:Proteins; D026403:S-Nitrosothiols; D013438:Sulfhydryl Compounds; D009569:Nitric Oxide; C073734:sinapinic acid; D001710:Biotin; D004228:Dithionitrobenzoic Acid; D001205:Ascorbic Acid