Flavoprotein assay

Foster The WC-1 photoreceptor analysis is lovely. In the binding assay you generated your action spectrum from, I was amazed to see that you got sufficient resolution in it to see the absolutely characteristic double peaks that you d expect from a flavoprotein-like pigment. If you ever get a chance to replot those data, fit a flavoprotein nomogram to it. I m sure that it will fit perfectly. 

Table 3.2.5 Disorders detectable by the in vitro probe assay. ETF Electron transfer flavoprotein, MADD multiple acyl-CoA dehydrogenase deficiency...

Like the nicotinamide coenzymes (Fig. 13-15), the flavin nucleotides undergo a shift in a major absorption band on reduction. Flavoproteins that are fully reduced (two electrons accepted) generally have an absorption maximum near 360 nm. When partially reduced (one electron), they acquire another absorption maximum at about 450 nm when fully oxidized, the flavin has maxima at 370 and 440 nm. The intermediate radical form, reduced by one electron, has absorption maxima at 380, 480, 580, and 625 nm. These changes can be used to assay reactions involving a flavoprotein. 

Glutathione is discussed further in the section on selenium and glutathione in Chapter 10. The enzyme assay is conducted using glutathione reductase extracted from red blood cells with and without added FAD. Chmnic consumption of a diet deficient in riboflavin allows the continued synthesis of a variety of flavoproteins, but results in the accumulation of apoenzyme without its conversion to holoen-zyme. Addition of chemically pure FAD to a biological fluid containing apoenzyme results In the stimulation of enzyme activity because of the formation of the holoenzyme. It is this stimulation of enzyme activity that is used to determine vitamin status in humans. 

The yeast ADH is very sensitive to various metal ions 1.14 X 10 M Cu", 1.52 X 10 M Kg, and 2.28 X 10 Hg result in 50% inhibition of yeast ADH. Ferric iron and zinc ions were also found to inhibit, but much less so. Ferrous iron and manganous ions did not inhibit at all (von Euler and Adler, 1935). The copper inhibition could be reversed by tenfold excess concentrations of glutathione and cyanide (Wagner-Jaueregg and Moller, 1935). The copper inhibition was confirmed by Negelein and Wulff (1937). It must be kept in mind, however, that the assay system of von Euler and Adler measured oxidation rates of both the flavoproteins and ADH. 

Table 10. Effect of erythrocuprein on cytochrome-c reductase activity of flavoproteins. The cytochrome-c reductase activity was measured in air-equilibrated solutions containing 0.1 M pyrophosphate, pH 8.5, in the presence of 3.33 X 70-5 M cytochrome c and 10 fig bovine catalase. The concentration of erythrocuprein in this assay mixture was 0.62 fiM. The temperature was 25° (150)...

The enzyme is a flavoprotein, and is one of the enzymes of the citric add cycle. It may be used as a marker enzyme for mitochondria. Assay may be accomplished by utilising reduction of dichlorophenolindophenol at 600 nm [614]. 

This is a flavoprotein enzyme containing molybdenum, which catalyses the reaction responsible for the terminal stage of purine metabolism in tissue. It can also oxidise hypoxanthine, monomethyixanthines, and some.aldehydes. It may be assayed radio-chemically using labelled xanthine and separation of urate with the aid of Dowex 50 [654]. 

The isolated reductase (above) was used in assays to direct further fractionation experiments which culminated in the isolation of the physiological reducing system this turned out to be a previously unreci ized hydrogen transport system. This system, which connects ribonucleotide reductase to the NADPH-NADP+ system, was found to be a two-component system con.sisting of a small sulfhydryl protein, thioredoxin, and a flavoprotein, thioredoxin reductase. Thioredoxin is the reductant which specifically interacts with the ribonucleotide reductase. In the presence of catalytic amounts of thioredoxin, the thioredoxin reductase will link NADPH with the reduction of ribonucleoside diphosphates as follows ... 

Potato Phenol Oxidase. The studies of Kubowitz on the copper oxidase of potatoes were part of the efforts of Warburg s institute to find enzyme systems that could catalyze oxygen consumption coupled with substrate oxidation. The enzyme was purified on the basis of an assay involving transfer of electrons from pyridine nucleotides to o-quinone and from the resulting catechol to oxygen (I). The reduction of catalytic quantities of quinone was probably catalyzed by a flavoprotein present in the Zwischenferment preparation used to reduce TPN. In the presence of... 

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