Micromethods for determination of ammonia, urea, total nitrogen, uric acid, creatinine (and creatine), and allantoin

The success with which Krebs and Henseleit (1) employed Warburg’s method of surviving tissue slices in the problem of the formation of urea in the liver encouraged its use for a direct attack on other problems of nitrogen metabolism in animals

The oxidation-reduction potential of coenzyme I

The oxidation-reduction potential of cozymase (diphosphopyridine nucleotide) was calculated from the free energies of formation of aqueous d-alanine and d-glutamic acid based on thermal data, and the equilibria measured by Wurmser and Filitti-Wurmser(1) for pyruvate + 2H+ + 2(e) ⇌ alanine + H2O, by Cohen(2) for α-ketoglutarate + alanine ⇌ d-glutamate and pyruvate, and by von Euler et al.(3) for the reaction α-ketoglutarate + NH+4 + reduced cozymase ⇌ glutamate + oxidized cozymase. The value for the potential so calculated is at 30° E’0 = -0.072 - 0.03 pH ± 0.0008 volt

The conversion of L-histidine to glutamic acid by liver enzymes

Edlbacher and Neber (1) showed in 1934 that the liver enzyme named histidase degrades histidine to NH3, formic acid, and an unknown product which on further treatment with strong alkali yields glutamic acid. This led to the suggestion that glutamic acid is a metabolic product of histidine, a suggestion that was supported by the finding that glycogen was formed from histidine about as well as from glutamic acid (2). These findings did not prove that glutamic acid was one of the products of histidine metabolism, and the idea became questionable when the evidence from subsequent investigations with non-isotopic histidine (3), imidazole-N16-histidine (4), and carboxyl-C14-histidine (5) were negative or inconclusive. In studies on the fate of carboxyl-C14-L-histidine in the liver of rabbits after injection and after incubation with guinea pig liver slices, we have found direct evidence that glutamic acid is a major product of histdine metabolism. Another highly radioactive compound was isolated by ion exchange chromatography, whose properties with respect to chromatography and lability to alkali and acid appear to correspond to those reported for isoglutamine. Takeuchi (6) isolated and identified isoglutamine as a product of the action of urocanicase on urocanic acid, which was obtained by the action of another liver enzyme on histidine. The formation of isoglutamine as an intermediate is consistent with our finding that the label in the radioactive glutamic acid formed from carboxyl-C14-histidine is not in the α-carboxyl group, and the inference is very strong that the label is in the γ-carboxyl group

The enzymatic synthesis of protein. II. The effect of temperature on the synthesizing action of pepsin

In a solution of the products of the hydrolysis of protein it is theoretically possible to bring about the reverse reaction, i.e. synthesis, in two ways: by concentrating the solution, and by raising the temperature. The theoretical considerations from which the first of these conclusions was deduced have been discussed in a previous paper (1). It is sufficient to recapitulate here, that the first method is predictable from an appropriate statement of the mass law. The experimental confirmation of the prediction was described by the authors (1). The second method is predictable from certain thermodynamical considerations of reversible reactions pointed out by Moore (2). He deduced the equilibrium equation Pα, = K Pnb, where Pα, and Pb, are respectively the osmotic pressures of the substrate and its product, and K is a constant. K is a symbol for the expression P0eC/RT, where P and e are constants, R is the gas constant, C is the chemical energy involved in the breakdown of 1 gram molecule of A into n gram molecules of B, and T is the absolute temperature

Nitrogen distribution by globin

This and other experiences with the tryptophane method of Fürth and Nobel led us to doubt seriously the reliability of quantitative data obtained by its application. When, therefore, just as we completed our work with it, Folin and Looney (6) described another and apparently better method of determination, a method based upon a different color reaction and capable moreover of convenient combination with a quantitative procedure for tyrosine, it seemed to us worth while to review the problem again. With the aid of this newer method we have now determined the tryptophane and tyrosine content of two series of globin preparations, and have, we believe, settled fairly decisively the proportion of these amino-acids yielded by the pure protein. We have also taken occasion to determine by the method of Van Slyke the general distribution of nitrogen in the globin molecule

The enzymatic synthesis of protein. IV. The effect of concentration on peptic synthesis

In the enzymatic hydrolysis and synthesis of proteins in vitro, the important factor, the factor upon which the direction and the degree of the reaction are dependent, is not the relative concentration of water, but the concentration of material in solution. This conclusion, pointed out by Moore, the authors have discussed at length in a previous paper (1). As shown there, the molecular concentration of water is always so enormously greater than that of the other components that the small amounts added or removed in the course of either reaction are negligible, and it may, therefore, be considered as remaining constant. The distinguishing feature of the hydrolysis and synthesis of protein is the conversion of 1 molecule of protein into a number of molecules of products. It is this characteristic which is responsible for complete hydrolysis in dilute solutions and for the ease with which synthesis is achieved in concentrated solutions. It follows that the extent of synthesis will increase as the concentration increases, and that as the concentration decreases a point will be reached at which synthesis will fail. The concentration at this point will correspond to the maximum concentration of protein capable of complete hydrolysis

A micromethod for the determination of glycocyamine in biological fluids and tissue extracts

In the two following communications (1, 2) evidence is presented that glycocyamine is a normal precursor of creatine in the animal body. These studies required a satisfactory micromethod for the determination of glycocyamine. The most reliable method described in the literature consists in adsorption on Lloyd’s reagent in acid solution, elution with baryta, removal of arginine from the eluate by repeated adsorption on permutit, and calorimetric determination of the remaining glycocyamine by means of the Sakaguchi reaction. There are only two substances which are common in biological fluids and which give an intense color in the Sakaguchi reaction. These are arginine and glycocyamine. This method was first introduced by Weber (3) and was modified by Bodansky (4) and by Davenport and Fisher (5). In our hands even the latest version of the method, that described by Davenport and Fisher, had the following shortcomings: it was laborious and time-consuming, the adsorption of the glycocyamine on the Lloyd’s reagent was incomplete, further losses of glycocyamine occurred in the repeated treatment with permutit (Davenport and Fisher report losing only 10 per cent in three adsorptions; with the permutit available to us we lost 80 per cent), and the color developed was unstable. Furthermore, the amount of glycocyamine lost on the permutit varied according to the amount of arginine present, the less arginine the greater the loss of glycocyamine. All these disadvantages have been removed in the method described below. It is the first method in which glycocyamine added to blood or urine can be determined quantitatively, even in concentration8 as low a8 0.1 mg. per cent. 2 to 5 ml. are sufficient for an analysis. An indication of the speed and convenience of the method is that twenty to forty analyses can be carried through simultaneously in about 2 hours

Dimethylthetin and dimethyl-β-propriothetin in methionine synthesis

In a previous communication it was shown that choline and betaine are effective in promoting methionine synthesis from homocysteine in tissue homogenates (1). Data presented in this paper indicate that dimethylthetin, (CH3)2+SCH2COO-, which has been shown by Welch (2) to be lipotropic and has been reported by du Vigneaud (3) to promote growth on a methionine-free, homocysteine-containing diet, is 20 times as active as betaine in methionine formation. Dimethyl-β-propiothetin, (CH3)2+S(CH2)2COO-, recently isolated from Polysiphonia fastigiata by Challenger and Simpson (4) is also highly active. The enzyme for this transmethylation is found in the liver and kidney of all animals tested. Its high activity and general distribution suggest its biological importance in methionine synthesis

The role of the enzyme in the succinate-enzyme-fumarate equilibrium

The following is an account of an investigation into the role of the enzyme in the succinate-enzyme-fumarate equilibrium. The method consisted in the comparison of the value of the free energy change in this reaction obtained from oxidation-reduction potentials, with that calculated from the entropies and other physicochemical properties of succinic acid and fumaric acid