Flavin chromatography

Hastings, J. W., Balny, C., Le Peuch, C., and Douzou, P. (1973). Spectral properties of an oxygenated luciferase-flavin intermediate isolated by low-temperature chromatography. Proc. Natl. Acad. Sci. USA 70 3468-3472. 

Flavin adenine dinucleotide Riboflavin 5 -monophosphate Riboflavin 5 -monophosphate, reduced form Fast protein liquid chromatography (Pharmacia)... 

C. Andres-Lacueva, F. Mattivi and D. Tonon, Determination of riboflavin, flavin mononucleotide and flavin-adenin dinucleotide in wine and other beverages by high-performance liquid chromatography with fluorescence detection. J. Chromatogr.A 823 (1998) 355-363. 

Flavin mononucleotide (Na, 2H2O salt, FMN) [130-40-5] M 514.4. Purified by paper chromatography using leri-butanol-water, cutting out the main spot and eluting with water. Also purified by adsorption onto an apo-flavodoxin column, followed by elution and freeze drying [Mayhew and Strating Eur J Biochem 59 539 1976]. 

The procedure to phosphorylate riboflavin derivatives on a preparative scale has recently been improved . These preparations, and also commercial FMN, contain a considerable amount of riboflavin phosphate isomers, which are difficult to separate by column chromatography. This problem is emphasized in the chemical synthesis of FAD where the yield is rather low (20-25 %). In this context, it is surprising that a modification of the synthesis of FAD from FMN published by Cramer and Neuhoeffer has not been noticed by workers in the flavin field. According to Cramer and Neuhoeffer, the yield of the chemical synthesis of FAD is drastically improved ( 70 % pure FAD). The procedure was successfully applied in the author s own laboratory (yield 60-70%). It is expected that the improved procedure of the FAD synthesis will stimulate the active-site directed studies on flavoproteins because the problem of separating FMN or FAD from their synthetic by-products has already been solved by use of FMN- or FAD-specific affinity column... 

Figure 3-5 Photograph of a two-dimensional thin layer (silica gel) chromatogram of a mixture of flavins formed by irradiation of 10 pg of the vitamin riboflavin. The photograph was made by the fluorescence of the compounds under ultraviolet light. Some riboflavin (RF) remains. The arrows indicate the location of the sample spot before chromatography. Chromatography solvents a mixture of acetic acid, 2-hutanone, methanol, and benzene in one direction and M-butanol, acetic acid, and water in the other. See Treadwell et al.H)2...

B Entsch, RG Sim. The purification and identification of flavin nucleotides by high-performance liquid chromatography. Anal Biochem 133 401-408, 1983. 

RP Hausinger, JF Honek, C Walsh. Separation of flavins and flavin analogs by high-performance liquid chromatography. Methods Enzymol 122G 199-209, 1986. 

Vastano et al. [8] have reported a method, based on solid phase extraction, with ion pair high performance liquid chromatography using fluorescence detection for the determination of flavins in seawater. Concentrations in the pm range could be determined. 

From the small subunit obtained by chaotrope-induced resolution of succinate dehydrogenase (143). The small subunit obtained by resolution and column chromatography in presence of SDS is completely free of flavin. 

By treatment with 5 M urea and chromatography on DEAE-cellulose, it has been possible to dissociate the E. coli NADPH-sulfite reductase into a flavoprotein and a hemoprotein fraction. The flavoprotein fraction has been shown to be an octamer of a single polypeptide of molecular weight 58,000-60,000 and to contain FMN and FAD in equimolar amounts, but no heme, nonheme iron, or labile sulfide. The hemoprotein fraction is a tetramer of a polypeptide of molecular weight 54,000-67,000, and contains heme, nonheme iron, and labile sulfide, but no flavin. Thus NADPH-sulfite reductase is considered to be an enzyme of agfit subunit composition. The amino acid composition of the whole enzyme and the flavoprotein and hemoprotein fractions have been determined (414). 

Fig. 43. Chromatography at -20° of luciferase-flavin intermediate on Sephadex LH-20. Ordinate absorbance at 280 (--), 370 (...), or 450 ( - , FMN) nm bioluminescence (--) (multiply by 5 x 1012 to obtain initial intensity in quanta sec-1 for a...

Intermediate IIA was prepared by the same method as intermediate II except that 0.1 mM octanal was present. Because aldehyde binding to luciferase is reversible and the two would presumably be separated in the column, octanal (50 pM) was also added to the column buffer (50% ethylene glycol phosphate). The activity was eluted in its characteristic position, but in smaller yield than intermediate II in the absence of aldehyde. This was partly due to the presence of a considerable amount of free luciferase. In the fractions eluted at the end, the ratio of flavin to protein increased, indicating that flavin initially bound to luciferase was released during chromatography and retarded somewhat on the column. This suggested that intermediate IIA was broken down on the column as the amount of FMN contaminating luciferase was considerable. It was therefore impossible to isolate pure intermediate IIA by this procedure. 

Purification of cytochrome 62 has been carried out by various workers S08Jill l4r-217). The cytochrome was first crystallized by Appleby and Morton in 1954 ( 806) as a preparation (type I) containing DNA (214,217). Another crystalline preparation (type II) which contains no DNA was also obtained after dialysis against strong ammonium sulfate solution or by chromatography on DEAE-cellulose (218-221). Another crystalline preparation, which is free from flavin and enzymically inactive, was obtained by Okunuki et al. (222-224). This cytochrome has a molecular weight of about 20,000 and appears to be similar to but different from the cytochrome bt core described later. 

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