Warburg and Christian

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. 

In 1931 Warburg and Christian (W5) detected an enzyme catalyzing the reduction of NADP by G-6-P, which he called Zwischenferment G-6-P + NADP+ 6-PG + NADPH + H+ (3)... 

Many of the amino acids originally tested by Krebs were racemic mixtures. When naturally occurring L-amino acids became available the oxidase was found to be sterically restricted to the unnatural, D series. [D-serine occurs in worms free and as D-phosphoryl lombricine (Ennor, 1959)]. It could not therefore be the enzyme used in the liver to release NH3 in amino acid metabolism. D-amino acid oxidase was shown by Warburg and Christian (1938) to be a flavoprotein with FAD as its prosthetic group. A few years later Green found an L-amino acid oxidase in liver. It was however limited in its specificity for amino acid substrates and not very active—characteristics which again precluded its central role in deamination. 

Determination of nucleic acid yield. Cleared lysate or ImuVert was diluted to a concentration which had an absorbance between 0.4 and 0.5 at 260 nm and the absorbances at 280 and 260 nm were measured. The nucleic acid concentration in solution was then calculated by the method of Warburg and Christian (18), The yield of nucleic acid was calculated by determining the percent of nucleic acid in the lysate that was isolated in the product. The acceptable range for ImuVert manufacturing is 10.5 + 1.5. 

According to Warburg and Christian the total amount of nucleic acids and protein is calculated using the following equation (the factors F and T are given in Table 1.10 in conjunction with the ratio A280/A260) ... 

Old yellow enzyme , first protein recognized Warburg and Christian 4)... 

The discoveries of Theorell and Warburg and Christian demonstrated that the prosthetic group of flavoenzymes can be released reversibly without proteolytic... 

Why do we need vitamins Early clues came in 1935 when nicotinamide was found in NAD+ by H. von Euler and associates and in NADP+ by Warburg and Christian. Two years later, K. Lohman and P. Schuster isolated pure cocarboxylase, a dialyz-able material required for decarboxylation of pyruvate by an enzyme from yeast. It was shown to be thiamin diphosphate (Fig. 15-3). Most of the water-soluble vitamins are converted into coenzymes or are covalently bound into active sites of enzymes. Some lipid-soluble vitamins have similar functions but others, such as vitamin D and some metabolites of vitamin A, act more like hormones, binding to receptors that control gene expression or other aspects of metabolism. 

Warburg and Christian recognized the relationship of NADP+ and NAD+ (then called DPN+) and proposed that both of these compounds act as hydrogen carriers through alternate reduction and oxidation of the pyridine ring. They showed that the coenzymes could be reduced either enzymatically or with sodium dithionite Na2S204. 

Nicotinic acid was prepared in 1867 by oxidation of nicotine. Although it was later isolated by Funk and independently by Suzuki in 1911-1912 from yeast and rice polishings, it was not recognized as a vitamin. Its biological significance was established in 1935 when nicotinamide was identified as a component of NAD+ by von Euler and associates and of NADP+ by Warburg and Christian.3 Both forms of the vitamin are stable, colorless compounds highly soluble in water. 

Warburg and Christian showed that the color of this old yellow enzyme came from a flavin and proposed that its cyclic reduction and reoxidation played a role in cellular oxidation. When NADP+ was isolated the proposal was extended to encompass a respiratory chain. The two hydrogen carriers NADP+ and flavin would work in sequence to link dehydrogenation of glucose to the iron-containing catalyst that interacted with oxygen. While we still do not know the physiological function of the old yellow enzyme,b the concept of respiratory chain was correct. 

Nucleic acids have substantial absorbance at 280 nm and can interfere with A2so quantitation of protein in crude samples. To resolve the protein concentration in such samples, measure the absorbance at 260 nm and 280 nm and calculate the protein concentration as follows (Warburg and Christian, 1942 Layne, 1957) protein concentration (mg/ml) = 1.55 x A2g0 -0.76 x A 260- This estimation of protein concentration is valid up to 20% (w/v) nucleic acid or an A2g )/A2(Jo ratio <0.6. 

Warburg and Christian discovered the yellow enzyme, a flavoprotein. 

In 1943 Warburg and Christian (W7) reported that five of the glycolytic enzymes are present in the blood serum of rats, and of these the activities of aldolase and triose isomerase are usually increased in the... 

Although crystalline riboflavin was not isolated until 1931 interest in this compound as a pigment dates back to 1881 in connection with the color in the whey of milk. In 19.31 riboflavin was isolated as a coenzyme-en/.yme complex from yeast by Warburg and Christian and was designated yeiioiv oxidation ferment. 

The reduction is effected by the oxidation of hexose-6-phosphate in presence of Warburg and Christian s Zwischenferment (1932). The initial esterification is brought about by the action of hexokinase on glucose and ATP. 

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