tert-Butylhydroquinone as a Spectroscopic Probe for the Superoxide Radical Scavenging Activity Assay of Biological Samples


Bekdeser B. , Ozyurek M. , Guclu K. , Apak R.

ANALYTICAL CHEMISTRY, vol.83, pp.5652-5660, 2011 (Journal Indexed in SCI) identifier identifier identifier

  • Publication Type: Article / Article
  • Volume: 83 Issue: 14
  • Publication Date: 2011
  • Doi Number: 10.1021/ac200788m
  • Title of Journal : ANALYTICAL CHEMISTRY
  • Page Numbers: pp.5652-5660

Abstract

As a more convenient and less costly alternative to electron spin resonance (ESR) and nonspecific nitroblue tetrazolium (NET) and cytochrome c assays of superoxide radical (SR, O-2(center dot-)) detection, a novel probe, tert-butylhydroquinone (TBHQ), is introduced for SR nonenzymatically generated in the phenazine methosulfate-beta-nicotinamide adenine dinucleotide (PMS-NADH) system. SR attacks both TBHQ and SR scavengers incubated in solution for 30 min where scavengers compete with TBHQ for the O-2(center dot-) produced. TBHQ but not its O-2(center dot-) product, tert-butyl-1,4-benzoquinone (TBBQ), is responsive to the CUPRAC (cupric reducing antioxidant capacity) spectrophotometric assay. The CUPRAC absorbance of the ethyl acetate extract of the incubation solution arising from the reduction of Cu(II)-neocuproine reagent by the remaining TBHQ was higher in the presence of O-2(center dot-) scavengers (due to less conversion to TBBQ), the difference being correlated to the SR scavenging activity (SRSA) of the analytes. With the use of this reaction, a kinetic approach was adopted to assess the SRSA of amino acids, vitamins, and plasma and thiol antioxidants. This assay, applicable to small-molecule antioxidants and tissue homogenates, proved to be efficient for cysteine, uric acid, and bilirubin, for which the widely used NBT test is nonresponsive. Thus, conventional problems of NBT assay arising from formazan insolubility and direct reduction of NBT by tested scavengers were overcome.