Flavins digestion

In contrast to the nicotinamide nucleotide dehydrogenases, the prosthetic groups FMN and FAD are firmly associated with the proteins, and the flavin groups are usually only separated from the apoen2yme (protein) by acid treatment in water. However, in several covalently bound flavoproteins, the enzyme and flavin coen2ymes are covalently affixed. In these cases, the flavin groups are isolated after the proteolytic digestion of the flavoproteins. 

NADPH-cytochrome P-460 reductase is composed of a single polypeptide chain of 70,000-80,000 molecular weight 369, 371, 373, 374) associated with one molecule of FAD and one molecule of FMN 370, 371, 373, 378, 379). These results apply to the enzyme whether solubilized by proteolytic digestion or by detergent extraction. The minimum molecular weight based on the flavin content is somewhat higher, 87,000 (373), possibly indicative of the flavin lability observed upon irradiation in ammonium sulfate 380). The detergent-solubilized reductase has a lower flavin content, 0.64 and 0.79 moles of FMN and FAD per 79,000 g of enzyme 371). The absorbance ratio, 275 nm 455 nm of 6.5, indicates a relatively low content of aromatic amino acids 373, 374) The extinction of the flavins at 455 nm is 10.7 mM cm" (373). 

As a result of collaborative work among the laboratories of Ehren-berg, Hemmerich, and Singer 161-163), the structure of the bound flavin was elucidated to be 8 -(A(-3-histidyl) flavin adenine dinucleotide. The structure of the histidyl flavin and the sequence of a flavin-bound penta-peptide isolated by proteolytic digestion are shown in Fig. 28. Singer et al. 23,25) have discussed the details of these studies. 

The existence of choline dehydrogenase was first demonstrated by Mann and Quastel in 1937 247, 248) in extracts of rat liver and kidney. These authors also obtained evidence that the first oxidation product of choline was betaine aldehyde. Others showed subsequently that choline oxidase activity resided in the mitochondrial fraction of rat liver and is linked to the respiratory chain 249, 250). Detergents 251, 252), solvent treatment of fragmented mitochondria 253), and venom phospholipase 254-256) have been used for extraction and solubilization of choline dehydrogenase. Among these, the best method reported to date appears to be the digestion of acetone-powdered mitochondria with venom phospholipase. Choline dehydrogenase, partially purified from phospholipase extracts of rat liver mitochondria, contains 1 mole of flavin and 4 g-atoms of nonheme iron per 850,000 g protein. The flavin is claimed to be acid-... 

The most common forms of vitamin B2 are riboflavin 5 -phosphate (FMN) and flavin adenine dinucleotide (FAD), which are best known for their participation as co-factors (ligands) to some of the enzymes involved in electron transfer chains. The ligand is usually coupled to enzymes through the phosphate moiety and therefore isolation from tissue can be achieved by either mild acid hydrolysis or by enzymatic digestion with an acid phosphatase. 

Azoreductase activity (substrate l,2-dimethyl-4-(/ -carboxyphenylazo)-5-hydroxybenzene (CPA) (Hanzel and Carlson 1974) and nitroreductase activity (substrate. 77-nitrobenzoic acid) (Carlson 1972) have been detected in digestive gland of M. mercenaria (Table 12). Their properties were similar to those of mammalian enzyme activities and considered indicative of the existence of two separate enzyme systems. Nitroreductase activity was maximal at 35 to 45 C and pH 6.0, Stimulated by flavin mononucleotide, and inhibited by potassium cyanide and air, but not by SKF-525A or carbon monoxide (cytochrome P-450 inhibitors). Azoreductase activity was similarly stimulated by flavin mononucleotide and inhibited by air, but had a pH optimum of 8.0 activity was higher at 37 C than at 22 °C. Both enzyme activities had cytosolic and microsomal subcellular localizations, viz. activity in nmol min g wet weight (% distribution in brackets)... 

Riboflavin needs to be present in the human typical diet, as animals, unlike many plants, fungi and bacteria, are unable to synthesize this molecule. Dietary intake of this vitamin includes free riboflavin and also its protein bound form, as FAD and FMN in flavoproteins (Figure 37.1 A). In the latter case, flavins need to be first released from carrier proteins during digestion and then hydrolysed to riboflavin by alkaline phosphatases and FMN/FAD pyrophosphatase in order to be absorbed at the small intestine. 

Figure 37.1 Riboflavin metabolism and cellular processing pathways. (A) Riboflavin and flavin intake is made via the diet, either in riboflavin-rich aliments or flavoproteins. In the latter, digestion in the stomach releases FAD and FMN cofactors. Riboflavin and flavins achieve a high concentration in the liver, spleen and cardiac muscle a concentration of about 30 nM riboflavin is also reached in the plasma circulation. (B) Riboflavin is imported into the cell and into the mitochondria via specific transporters (white circles in membranes). In the cytoplasm, flavin kinase (FK) and FAD synthetase (FADS) consecutively convert riboflavin into FMN and FAD, at the expense of ATP. An identical mechanism is also thought to be present inside the mitochondria, although a mitochondrial FK remains to be identified. FAD can also be imported into the mitochondria, or diffuse passively when the riboflavin concentrations are high. Figures reprinted from Henriques et al. (2010), with permission.

An extraction protocol, analogous to that described for vitamin Bi, permits measuring all flavins, both the endogenous ones and those used as supplements [1,6,8,10] acidic hydrolysis promotes the release of the protein-boimd forms and converts FAD to FMN the succeeding enzymatic digestion (takadiastase, amylase, acid phosphatase, claradiastase) is used for dephosphorylating FMN and hydrolyzing starch. 

As there was no information on the absorption, metabolism, and excretion of 8a-(amino acid)riboflavins, which arise through digestion and by turnover of flavoproteins that contain them, studies were made on the S-cysteinyl-flavin in rats. s The flavin moiety was traced by radiolabeling the relatively stable 2-position in the isoalloxazine. The cysteinyl portion was N-acetylated to prevent the too facile sulfoxidation that occurs nonenzymatically in aerobic, aqueous solutions of the flavinyl compound with a free a-amino function. 

Schabort and Potgieter (1971) showed that each of the J -cyclopiazonate oxidocyclases contain one covalently linked molecule of flavin per molecule of enzyme. The purified enzymes had a yellow color, and their ultraviolet and visible spectra showed maxima at 276, 366, and 450 nm. The addition of sodium dithionite or of jSCA caused the disappearance of the 450 nm peak because of the reduction of the flavin moiety. The flavin residues released from the protein molecules by proteolytic digestion with pronase showed absorption peaks at 262, 370, and 450 nm. The released flavin residues, separated by paper chromatography, were found to contain a covalently bound amino acid or small peptide. It was concluded that in the native enzyme, the flavin was bound by covalent linkage to the protein and that, during digestion with pronase, proteolysis stops at points determined by the specificity of the pronase or by structural or steric hindrance by the flavin. Pentose determination on the flavin residues showed that the flavins were dinucleosides. 

In subsequent studies (Kenney et ai, 1974a, 1976a) of the flavin prostjietic group of the j -cyclopiazonate oxidocyclases, the isoenzymes were digested with trypsin and chymotrypsin. A flavin mononucleotide peptide was isolated from the resultant mixture by chromatography on Florisil and diethylaminoethyl cellulose and by hydrolysis with nucleotide pyrophosphatase. Its amino acid composition was determined after hydrolysis in... 

The 1,8- and 4a,5-isomers are hitherto only known as 8- (181, 182) or 4a-alkyl-derivatives (13). The alkyl groups migrate intramolecularly between these positions and position 5, depending on pH, (55 8). Active residues such as benzyl and benzyl homologs C6H5CR2- (R = H or CH3) reach isomerization equilibrium within seconds at room temperature. This demonstrates that flavin is an active surface which is capable of digesting a-pairs as well as single or paired n-electrons, at specific sites (cf. Scheme 5). 

As in the case of many other types of natural products, the structural elucidation of the first member of this new class, the coenzyme of succinate dehydrogenase (SD-flavin), proved to be the most arduous and time-consuming (see introduction). Following Kearney s early work with SD-flavin peptides. Singer s group obtained on digestion with... 

In the last few years a further type of modified flavocoenzyme has beeen discovered which is structurally related to the coenzymes of succinate dehydrogenase and monoamine oxidase, but differs considerably in its chemical properties. Early literature reports indicated the presence of a flavin in cytochrome C552 from Chromatium which could not be extracted with trichloroacetic acid or acidic ammonium sulfate (7), but could be released, for example by trypsin digestion or by incubation with saturated urea solutions (2). Absorption, fluorescence and ESR behaviour were closely similar to those of 8a-cysteinyl-ribo-flavin (12) and indicated the presence of a covalent link to the protein, through position 8a (185). Strong acid hydrolysis of these peptides liberated the flavin as mixture of riboflavin derivatives oxidation with per-formic acid and acid dephosphorylation yielded a homogeneous riboflavin derivative, which was identical with 8-nor-8-carboxy-riboflavin (185). 

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