Riboflavin isolation

Two types of fluorescent proteins have been isolated from luminous bacteria and studied in detail. The first of them are the blue fluorescent lumazine proteins (LumPs) containing lumazine as their chromophores, which were isolated from P. phosphoreum and P. fischeri (Gast and Lee, 1978 Koda and Lee, 1979 O Kane et al.y 1985). The second are the yellow fluorescent proteins (YFPs) containing a chromophore of FMN or riboflavin, isolated from P. fischeri strain Y-l (Daubner et al., 1987 Macheroux et ai, 1987 ... 

It was explained above how an antipellagra factor was included in the vitamin B2 complex. However, it was demonstrated that vitamin B2 (riboflavin), isolated from the vitamin B2 complex, did not show antipellagra activity in rats. As a result, the antipellagra factor separated from the vitamin B2 complex was called vitamin Bg [5]. Consequently, the factor that cures human pellagra is clearly different from this vitamin Bg. Thus, as the cure factors for black tongue disease of the dog, which resembles human pellagra, nicotinic acid and nicotinamide were isolated. 

An obsolete name for riboflavin (vitamin B-2) which described the origin of the isolate in the early days of riboflavin research. Hence, riboflavin isolated from milk was called... 

In 1933, R. Kuhn and his co-workers first isolated riboflavin from eggs in a pure, crystalline state (1), named it ovoflavin, and deterrnined its function as a vitamin (2). At the same time, impure crystalline preparations of riboflavin were isolated from whey and named lyochrome and, later, lactoflavin. Soon thereafter, P. Karrer and his co-workers isolated riboflavin from a wide variety of animal organs and vegetable sources and named it hepatoflavin (3). Ovoflavin from egg, lactoflavin from milk, and hepatoflavin from Hver were aU. subsequently identified as riboflavin. The discovery of the yeUow en2yme by Warburg and Christian in 1932 and their description of lumiflavin (4), a photochemical degradation product of riboflavin, were of great use for the elucidation of the chemical stmcture of riboflavin by Kuhn and his co-workers (5). The stmcture was confirmed in 1935 by the synthesis by Karrer and his co-workers (6), and Kuhn and his co-workers (7). 

Flavin mononucleotide was first isolated from the yellow en2yme in yeast by Warburg and Christian in 1932 (4). The yellow en2yme was spHt into the protein and the yellow prosthetic group (coen2yme) by dialysis under acidic conditions. Flavin mononucleotide was isolated as its crystalline calcium salt and shown to be riboflavin-5Lphosphate its stmeture was confirmed by chemical synthesis by Kuhn and Rudy (94). It is commercially available as the monosodium salt dihydrate [6184-17 /, with a water solubiUty of more than 200 times that of riboflavin. It has wide appHcation in multivitamin and B-complex solutions, where it does not require the solubili2ers needed for riboflavin. 

Riboflavin-5 -Adenosine Diphosphate. Riboflavin-5 -adenosine diphosphate [146-14-5] (flavin—adenine dinucleotide, FAD), C27H33N9O15P2 (2), mol wt 785.56, was first isolated in 1938 from the D-amino acid oxidase as its prosthetic group (95), where it was postulated to be... 

Biotransformations are carried out by either whole cells (microbial, plant, or animal) or by isolated enzymes. Both methods have advantages and disadvantages. In general, multistep transformations, such as hydroxylations of steroids, or the synthesis of amino acids, riboflavin, vitamins, and alkaloids that require the presence of several enzymes and cofactors are carried out by whole cells. Simple one- or two-step transformations, on the other hand, are usually carried out by isolated enzymes. Compared to fermentations, enzymatic reactions have a number of advantages including simple instmmentation reduced side reactions, easy control, and product isolation. 

Riboflavin was first isolated from whey in 1879 by Blyth, and the structure was determined by Kuhn and coworkers in 1933. For the structure determination, this group isolated 30 mg of pure riboflavin from the whites of about 10,000 eggs. The discovery of the actions of riboflavin in biological systems arose from the work of Otto Warburg in Germany and Hugo Theorell in Sweden, both of whom identified yellow substances bound to a yeast enzyme involved in the oxidation of pyridine nucleotides. Theorell showed that riboflavin 5 -phosphate was the source of the yellow color in this old yellow enzyme. By 1938, Warburg had identified FAD, the second common form of riboflavin, as the coenzyme in D-amino acid oxidase, another yellow protein. Riboflavin deficiencies are not at all common. Humans require only about 2 mg per day, and the vitamin is prevalent in many foods. This vitamin... 

Harkness DR, L Tsai, ER Stadtman (1964) Bacterial degradation of riboflavin V. Stoichiometry of riboflavin degradation to oxamide and other products, oxidation of C " -labeled intermediates and isolation of the pseudomonad effecting these transformations. Arch Biochem Biophys 108 323-333. 

A number of nitrogen heterocyclic, aromatic compounds, riboflavin 26, folic acid 27a and biopterin 27b, isolated from natural sources, are related in structure to natural redox enzyme cofactors. The electrochemistry of these and related compounds has been studied extensively. 

More than one century ago a yellow, fluorescent pigment was isolated from whey by Blyth In the subsequent years yellow pigments were extracted from various biological materials. Depending either on the source of isolation or the physical appearance, these natural products were named e.g. lactochrome , lycochrome , ovoflavin , lactoflavin , hepatoflavin , or verdoflavin . Later, it became evident that all these compounds are riboflavin (vitamin B2). 

Another Japanese research group isolated two riboflavin derivatives from the culture filtrate of Schizophyllum commune and called these natural products Schizo-flavin 1 and 2 The physical and chemical properties of these compounds are... 

The bright orange-yellow color and brilliant greenish fluorescence of riboflavin first attracted the attention of chemists. Blyth isolated the vitamin from whey in 1879 and others later obtained the same fluorescent, yellow compound from eggs, muscle, and urine. All of these substances, referred to as flavins because of their yellow color, were eventually recognized as identical. The structure of riboflavin was established in 1933 by R. Kuhn and associates, who had isolated 30 mg of the pure material from 30 kg of dried albumin from 10,000 eggs. The intense fluorescence assisted in the final stages of purification. The vitamin was synthesized in 1935 by R Karrer.3... 

Mitochondrial succinate dehydrogenase, which catalyzes the reaction of Eq. 15-21, contains a flavin prosthetic group that is covalently attached to a histidine side chain. This modified FAD was isolated and identified as 8a-(Ne2-histidyl)-FAD 219 The same prosthetic group has also been found in several other dehydrogenases.220 It was the first identified member of a series of modified FAD or riboflavin 5 -phosphate derivatives that are attached by covalent bonds to the active sites of more than 20 different enzymes.219... 

Many ascidians live gregariously, and for some of these species conspecific chemical cues may play an important role in gregarious settlement of the larvae. The extracts of conspecific adults, larvae, or their conditioned seawater have been shown to contain metamorphosis inducers which have never been characterized.74-75 Recently, Fusetani and co-workers elucidated the structure of the metamorphosis inducer for the solitary ascidian Halocynthia roretzi, which was isolated from seawater conditioned by ascidian larvae.10 The compound isolated from the medium was identical to lumichrome, a compound known to be a degradation product of riboflavin (Figure 13.1). The origin of lumichrome in //. roretzi is not known at present. 

H202 formation by tylakoids or isolated PSI was followed in a transparent lucite cuvette as previously described (De la Rosa et al., 1986 Navarro et al., 1987a). Riboflavin at 10 pM final concentration was used as the redox catalyst. Thylakoid and PSI suspensions were prepared from spinach bought in the market following the procedures of Amon and Chain (1977) and Peters et al. (1983), respectively. 

Walker and Nicholas (338) have reported the isolation and 600-fold purification of an enzyme from P. aeruginosa, which reduces nitrite to nitric oxide. The preparation contained 1.5 nmoles of FAD per mg protein, a c-type cytochrome and an absorption band at 630-635 nm, suggestive of copper. As electron donors, reduced FMN, FAD, riboflavin, pyo-cyanine, and methylene blue were effective, but not NADH, NADPH, or reduced cytochrome c. The preparation required phosphate or sulfate for maximal activity. The cytochrome and the 630-635-nm band were reduced under anaerobic conditions with a suitable electron donor and readily oxidized by nitrite. The K for NaNOj is reported to be 3.1 X 10 ... 

Most tissues contain very little free riboflavin and, except in the kidneys, where 30% is as riboflavin phosphate, more than 80% is FAD, almost all bound to enzymes. Isolated hepatocytes (and presumably other tissues) show saturable concentrative uptake of riboflavin. The of the uptake process is the same as that of flavokinase, and uptake is inhibited by inhibitors of flavokinase, suggesting that tissue uptake is the result of carrier-mediated diffusion, fol-lowedbymetabolic trapping as riboflavin phosphate, then onward metabolism to FAD, catalyzed by FAD pyrophosphorylase. FAD is a potent inhibitor of the pyrophosphorylase and acts to limit its own synthesis. FAD, which is not protein bound is rapidly hydrolyzed to riboflavin phosphate by nucleotide pyrophosphatase unbound riboflavin phosphate is similarly rapidly hydrolyzed to riboflavin by nonspecific phosphatases (Aw et al., 1983 Yamada et al., 1990). 

However, even relatively low concentrations of riboflavin can cause damage to DNA under conditions of photolysis, with damage to deoxy-guanosine in isolated DNA, and activation of DNA repair mechanisms in cells in culmre. it is therefore not common practice to use riboflavin supplements as an adjunct... 

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