Flavoprotein measurement

To study the effect of the protease treatment cell-free suspension, with or without protease treatment, was subjected to gel-filtration chromatography on Sephadex G-75 and the elution patterns were compared (Fig. 1). In each case, two major peaks were detected by monitoring column fractions with absorbance at 280 nm. Degradation activities on mexacarbate, in the presence of FMN and light under anaerobic condition, were measured for each fraction. It was found that the highest activity was associated with peak II. It is interesting to note that protein (s) associated with peak II were detected with or without protease treatment these will be referred to as natural flavoprotein (B, Fig. 

Porras, A. G., Palmer, G. Room temperature potentiometric measurements on xanthine oxidase. In Flavins and flavoproteins (Massey, V., Williams, C. H. eds.) pp. 810-820, New York, Amsterdam, Oxford, Elsevier/North Holland 1982... 

The fluorescence intensity and anisotropy of the flavin of a flavoprotein have been measured from 0 to 4 kbar. We notice an increase in the fluorescence intensity and a decrease in the fluorescence anisotropy. Explain the results in five to seven lines. 

Fortunately, the characteristic absorbance of certain stable and transient enzyme species and, in some instances, of products, together with the fact that the two half-reactions can be studied separately, permits informative rapid kinetic measurements of the overall and partial reactions of flavoprotein oxidases. Stopped-flow spectrophotometric methods (26) have been particularly useful (the irreversibility of the partial and overall reactions rules out relaxation methods) because the measured rate constants often correspond in part or whole to the reciprocals of the steady state coefficients. This is the major reason for using the formulation... 

The expected result was obtained since n-amino acid oxidase converted )8-chloroalanine to pyruvate under anaerobic conditions, to chloropyruvate at high O2 concentrations, and to mixtures of these at intermediate O2 concentrations. Under steady state conditions, the reaction behaved as if cleavage of the a C-H bond were the rate-limiting process in turnover, although stopped-flow spectrophotometric measurements showed that this interpretation can not be entirely correct in this case (53) or in the case of )8-choloro-a-aminobutyrate (54), a-yS-Elimination has now been observed in three flavoprotein oxidase reactions (54) and can be considered strong circumstantial evidence for a-proton removal from compounds which closely resemble the physiological substrates. 

Heering, H.A., Weiner, J.H., and Armstrong, F.A. (1997) Direct detection and measurement of electron relays in a multicentered enzyme voltammetry of electrode-surface films of E. coli fumarate reductase, an iron-sulfur flavoprotein. Journal of the American Chemical Society, 119,11628-11638. 

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. 

Lactate monooxygenase (LMO, EC 1.13.12.4) from Mycobacterium smegmatis is a flavoprotein with a molecular weight of 340 000 and a -KMflactate) of 8 mmol/1. The enzyme has been immobilized on porous glass and employed in an enzyme thermistor allowing lactate to be measured in the range 0.005-2 mmolA (Danielsson et al., 1981). 

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)...

Most of the microsomal reactions can be classified as oxidations by what are referred to as mixed-function oxidases utilizing molecular oxygen and cofactors. The key enzyme is an iron-hemecytochrome P-450, a flavoprotein dependent in its reduction and reoxidation on the NADPH to NADP reaction. The 450 notation is based on the 450 nm absorption peak the enzyme exhibits on reaction with carbon monoxide. Thus, drug interactions with this enzyme system can be evaluated by measuring absorption spectra changes. 

This review is divided into the following sections. First, EPR measurements of g-tensors of flavoproteins and the modulation of the principal values of g by the protein surroundings of the cofactor are discussed. Then, two recent examples of application of pulsed ENDOR spectroscopy will be reviewed, and, finally, time-resolved EPR spectroscopy, that is most favorably used to study photo-excited triplet states and radical pairs, will be introduced. 

Fig. 7 Triplet and radical-pair TREPR spectra of flavoproteins. (a) TREPR data set of the photoexcited triplet state of FMN in frozen aqueous solution measured 1 ps after pulsed laser excitation at 80 K [93].

A significant advance in understanding of the mechanism of the dihydrolipoic dehydrogenase reaction resulted from the discovery by Massey (1958) that the classic flavoprotein first isolated by Straub (1939), and widely known as Straub s diaphorase, behaves as a powerful dihydrolipoic dehydrogenase. Diaphorase activity was measured with ferricyanide as electron acceptor, Eq. (27), and dihydrolipoic dehydrogenase activity by... 

It had been suggested by Franke and Deffner that this enzyme was a flavoprotein. Notatin has absorption bands at 377 and 455 m, and the prosthetic group was identified as FAD. However, the process of splitting the enzyme appears to denature the protein, and it has not been possible to establish that FAD will restore the catalytic activity. The molecular weight has been determined by sedimentation measurements to be 152,000,... 

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