P-Mercuriphenyl sulfonate

Expressed as micromoles of NADH oxidized per min per mg of protein at 38°. All activities are completely inhibited with 0.1 mM p-mercuriphenyl sulfonate. 

D-Lactate cytochrome c reductase is inhibited by p-mercuriphenyl sulfonate salts, metal chelators, and dicarboxylic acids such as oxalate and oxaloacetate (Table XVI) (312, 314, 315). According to Nygaard (314), salts (cations) inhibit at the acceptor site, and dicarboxylic acids at the substrate site. Cremona and Singer (315) have studied the inhibitions by metal chelators and by oxalate. They recognized two types of inhibition. One type of inhibition is that which is caused by EDTA or oxalate. This kind of inhibition is reversed immediately upon dilution of the enzyme-inhibitor mixture. The second is that which results from addition of o-phenanthroline. Enzyme preparations treated with o-phenanthroline bind 2 moles of the chelator per mole of Zn . This complex is stable and inactive, and does not result in the release of Zri . The inactive... 

Treatment of E. coli sulfite reductase with p-mercuriphenyl sulfonate results in the specific release of FMN from the enzyme (390). FMN-depleted sulfite reductase can be prepared also by photodestruction of FMN. The enzyme-FMN dissociation constant is 10 nAf at 25°, and light irradiation can deplete the enzyme of FMN by destroying the released flavin. These treatments do not lead to removal or destruction of other components of the enzyme. The FMN-depleted enzyme is no longer capable of NADPH-dependent reduction of sulfite, nitrite, hydroxylamine. 

Inhibition by a variety of metal-binding agents competitive with respect to phosphoryl substrates (118-120) has suggested that an enzyme-bound divalent cation (other than Mg2+) may participate also in the binding of phosphate substrates. Observed inhibition by p-chloro-mercuriphenyl sulfonate and iodoacetate suggests the possibility that sulfhydryl groups may also be involved at, or near, the active enzymic site (119, 120). 

Searls and Sanadi (1959, 1960a) and Massey et al. (1960) observed an increase in absorbancy in the region between 500 and 600 mju (maximum at 530 m/i), concomitant with a decrease at 455 mju, on reduction of pig heart dihydrolipoic dehydrogenase with DPNH and with dihydrolipoic acid.A similar effect was noted previously by Savage (1957) on reduction of Straub s diaphorase with DPNH. These results recalled similar observations by Beinert (1957) with other flavoproteins which were attributed to the formation of a flavin semiquinone. Massey ei al. (1960) have made a detailed study of the stoichiometry of formation of the 530-mju band and the kinetics of its formation and disappearance under a variety of conditions, and attributed it to a flavin semiquinone which is an obligatory intermediate in the catalytic cycle of the enzyme. Addition of p-chloro-mercuriphenyl sulfonate to the partially reduced flavoprotein resulted in disappearance of the red color and further reduction of the flavin. This observation was interpreted as indicating that the flavin semiquinone is stabilized by interaction with a protein sulfhydryl group. 

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