The Fluorometric Measurement of the Nucleotides of Riboflavin and Their Concentration in Tissues

Proton transfer reactions of the oxidized NAD+-pyruvate adduct in its electronically excited singlet state were studied in aqueous solution and when bound to several proteins. The ionization constant of the adduct in the excited state was found to differ by several orders of magnitude from its value in the ground state. A rapid deprotonation reaction was found to follow electronic excitation if a suitable proton acceptor is present. The deprotonation is accompanied by a marked change in the fluorescence spectrum of the adduct and is therefore easy to detect and follow. The free adduct in aqueous solution was found to be in equilibrium between open and folded conformations of which only the open one is fluorescent. The equilibrium constant between these two forms was evaluated. A complex dependence of the fluorescence on the pH in the range of 1--7 was found, which originates in a shift of the equilibrium constant toward the open conformation and in protonation of acid groups of the adduct. Interaction of the adduct with four proteins was studied. Each had a different effect on the proton transfer reaction, reflecting differences in the microenvironment of the bound adduct. A possible use of the adduct as an excited-state proton transfer probe for proteins is presented.

Section: Discussionsupporting

confidence: 88%

Acid-base equilibriums of the oxidized .beta.-nicotinamide adenine dinucleotide-pyruvate adduct in the ground and electronically excited states. A proton transfer probe for proteins

Gafni¹

1980

“…The released flavin was confirmed to be FAD, first, from its identical migration with authentic FAD on TLC in two solvent systems2 (see Materials and Methods) and, second, from an approximately 10-fold fluorescence enhancement upon adding snake venom phosphodiesterase (Bessey et al, 1949).…”

Section: Resultsmentioning

confidence: 92%

Purification and properties of the flavoenzyme D-lactate dehydrogenase from Megasphaera elsdenii

Olson¹,

Massey²

1979

A pyridine nucleotide independent D-lactate dehydrogenase has been purified to apparent homogeneity from the anaerobic bacterium Megasphaera elsdenii. The enzyme has a molecular weight of 105 000 by sedimentation equilibrium analysis with a subunit molecular weight of 55 000 by sodium dodecyl sulfate gel electrophoresis and is thus probably a dimer of identical subunits. It contains approximately 1 mol of FAD and 1 g-atom of Zn2+ per mol of protein subunit, and the flavin exhibits a fluorescence 1.7 times that of free FAD. An earlier purification [Brockman, H. L., & Wood, W. A. (1975 J. Bacteriol. 124, 1454--1461] results in substantial loss of the enzyme's zinc, which is required for catalytic activity. The new purification yields greater than 5 times the amount of enzyme previously isolated. The enzyme is specific for D-lactate, and no inhibition is observed with L-lactate. Surprisingly, the enzyme has a significant oxidase activity, which depends on the ionic strength. Vmax values of 190 and 530 min-1 were obtained at a gamma/2 of 0.224 and 0.442, respectively. Except for this atypically high oxygen reactivity, D-lactate dehydrogenase resembles other flavoenzyme dehydrogenases in that the flavin does not react with sulfite, the tryptophan content is low, and a neutral blue semiquinone is formed upon photochemical reduction. The enzyme flavin is reduced either by dithionite, by oxalate plus catalytic 5-deazaflavin in the presence of light, or by D-lactate. Two electrons per flavin were consumed in a dithionite titration, implyine with varying ratios of D-lactate and pyruvate, an Em7 of -0.219 +/- 0.007 V at 20 degrees C was calculated for the flavin. The enzyme requires dithiothreitol for stability. Rapid inactivation results when the enzyme is incubated with a substoichiometric level of Cu2+. This inactivation can be reversed by dithiothreitol. It is proposed that the enzyme possesses a pair of cysteine residues capable of facile disulfide formation.

“…This approach to molecular-weight estimation was determined in other work to be valid for cytochrome P-450 reductase by the linearity of a Ferguson plot (Ferguson, 1964) of the reductase at various gel concentrations and the identity of the relative free mobility of the reductase with that of the marker proteins (Knapp et al, 1976). The minimum subunit molecular weight based on flavin content, determined by the procedure of Bessey et al (1949), and on protein concentration determined according to Lowry et al (1951), is 74 100. A minimum molecular weight of about 76 500, detemined in sedimentation equilibrium studies of the reductase in 6 M guanidine hydrochloride, is in agreement with these other estimations.…”

Section: Resultsmentioning

confidence: 99%

NADPH-cytochrome P-450 reductase from rat liver: purification by affinity chromatography and characterization

Dignam¹,

Strobel²

1977

209

(NADPH)-cytochrome P-450 reductase was purified to apparent homogeneity by a procedure utilizing nicotinamide adenine dinucleotide phosphate (NADP)-Sepharose affinity column chromatography. The purified flavoprotein has a molecular weight of 79 700 and catalyzes cytochrome P-450 dependent drug metabolism, as well as reduction of exogenous electron acceptors. Aerobic titration of cytochrome P-450 reductase with NADPH indicates that an air-stable reduced form of the enzyme is generated by the addition of 0.5 mol of NADPH per mole of flavin, as judged by spectral characteristics. Further addition of NADPH causes no other changes in the absorbance spectrum. A Km value for NADPH of 5 micron was observed when either cytochrome P-450 or cytochrome c was employed as electron acceptor. A Km value of 8 +/- 2 micron was determined for cytochrome c and a Km of 0.09 +/- 0.01 micron was estimated for cytochrome P-450.