The biological applications of tetrazolium salts are the subject of a textbook.96 Kuhn and Jerchel74 were the first to recognize the utility of tetrazolium salts as indicators in redox enzyme activity, particularly those of the various dehydrogenases. It has been recognized449 that this particular utility of tetrazolium salts is related to the proximity of their redox potentials to those of the hydride transfer systems in biology450 such as nicotinamide adenine dinucleotide, NAD, and its phosphate analogue, NADP.

The reduction of tetrazolium salts by NADH is greatly accelerated by electron transfer agents (ETAs) such as phenazine methosulfate (PMS; 233) or its derivatives.451-454 Other classes of ETAs such as quinones.455,456 ferricinium,457 phenothiazine,458 the viologens,459 acridiniums,460 and phe — nazinium or quinoxalinium salts461 as well as the enzyme diaphorase462 have been used.

The mechanism of this electron transfer has been the subject of many studies. Many workers support the involvement of the superoxide radical ion.463-468 However, a recent study469 based on EPR470 and electrochemi-







MPH = Reduced Phenazinium FH = Formazan T+ = Tetrazolium

Scheme 35

cal data471 concludes that the superoxide radical, while capable of interac­ting with both the tetrazolium salt and PMS, is not involved in the redox cycle NADH/tetrazolium (Scheme 35).

Tetrazolium salts are used to follow the reaction of these enzymes or to quantify their substrates/products. Therefore, a variety of tetrazol — ium salts have been used to study a large number of dehydrogen- ases.439,440,475-491 Other enzymes that generate products that are susceptible to oxidation by dehydrogenases can also be followed with tetrazolium salts.494 For example, indoxyl derivatives (234) can act as substrates for hydrolytic enzymes such as phosphatase and galactosidase. The resulting indoxyl (235) reduces tetrazolium salts to formazans providing a method for the determination of these enzymes (Eq. 29).492-496 Since redox enzymes, including dehydrogenases, are widely spread in living systems, tetrazolium salts as direct or indirect detectors of these activities have found use in all areas of life sciences; only some aspects of these applications are discussed here.





x = OPO3H2


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