Photoaffinity inhibition of rat liver NAD(P)H dehydrogenase by 3-(.alpha.-acetonyl-p-azidobenzyl)-4-hydroxycoumarin

Abstract

NAD(P)H dehydrogenase was purified in four steps from a homogenate of rat liver. The final step was affinity chromatography on Sepharose coupled to 3,3'-(m-hydroxybenzylidene)bis(4-hydroxycoumarin). The purified enzyme was inhibited competitively with respect to NADH by 3-(alpha-acetonyl-p-nitrobenzyl)-4-hydroxycoumarin (acenocoumarin) (Ki = 1.7 microM). The acenocoumarin was converted into an azide which was used to photoaffinity inhibit the enzyme. Following photolysis in the presence of the azide, the enzyme was inactivated in proportion to the concentration of azide present during irradiation. A maximum of 35-40% inhibition could be achieved by a single irradiation at 254 nm for 1.5 min. This inhibition was noncompetitive with respect to NADH. The inactivation was shown to be specific as acenocoumarin afforded complete protection against inactivation, irradiation was required to achieve inactivation, and the enzyme was unaffected by irradiation alone.

Synthetic Approaches and Biological Activities of 4-Hydroxycoumarin Derivatives

“…They also prepared various radiolabeled 3-substituted 4-hydroxycoumarin derivatives using commercially available [U- 14 C] phenol or [U- 3 H] in order to elucidate the binding photoaffinity. Compounds 62-64 serve as effective competitive inhibitors of the dicoumarol sensitive NADPH quinone reductase from rat liver [28,29]. …”