Silver-induced enhancement of thiochrome-based peroxide measurements

Thiamine is presently one of the most attractive substrates used for sensitive fluorometric measurements of peroxides. Thiochrome (TC), a highly fluorescent product, is formed in enzyme-mediated oxidations. It is assumed that H(2)O(2) is nearly quantitatively converted to TC. The reaction cannot differentiate H(2)O(2) from many other peroxides such as methylhydroperoxide (MHP); to perform differential measurements, H(2)O(2) can first be selectively destroyed by a suitable catalyst such as MnO(2). In substituting Ag(2)O for MnO(2) to accomplish the selective destruction of H(2)O(2), we achieved the stated objective but were puzzled by a 3-fold increase in the MHP response in the presence of Ag(2)O. It was soon discovered that traces of dissolved Ag(+) and Hg(2+) can dramatically increase the yield of TC in this reaction from either H(2)O(2) or MHP; the normal yield in fact is only 20%. We present here a reaction scheme and kinetic model that adequately describes this behavior and should provide a path to substantially increase the sensitivity of this important assay method.