Donald D. Van Slyke

The sulfhydryl nature of collagen proline hydroxylase

Abstract A partially purified preparation of collagen proline hydroxylase from chick embryos is strongly inhibited by sulfhydryl reagents such as N -ethylmaleimide and p -chloromercuribenzoate. The presence of α-ketoglutarate, one of the substrates of the enzyme, affords considerable protection against inhibition by N -ethylmaleimide. The activity of collagen proline hydroxylase is stimulated by the presence of bovine serum albumin in the assay mixture, as Rhoads, Hutton, and Udenfriend have shown. However, the effect consists of two parts. The larger of the two is found to be independent of the sulfhydryl content of the albumin, but a small amount of further stimulation can be abolished by pretreating the albumin with N -ethylmaleimide. Dithiothreitol, which was shown by Rhoads, Hutton, and Udenfriend to stimulate the enzyme when present at concentrations up to 0.3 m m , is found to destroy the enzymic activity at higher concentrations (0.5 or 1.0 m m ). It seems probable that both a sulfhydryl group and one or more disulfide links in the enzyme are necessary for its activity. The sulfhydryl group may be in or near the site for the binding of α-ketoglutaric acid.

RENAL TUBULAR FAILURE OF SHOCK AND NEPHRITIS

Excerpt The manner in which tubular injury can cause excretory failure was observed by A. N. Richards in 1929.1He watched through a microscope the nephrons of frogs that had been poisoned with subl...

Preparation of Blood Lipid Extracts Free from Non-Lipid Extractives:

Deficiencies in Purification of Lipids by Petrol Ether. Resolution in petrol ether has been a classical procedure for analytical purification of extracted fats. Thus the blood fats extracted with Bloors 1 efficient alcohol-ether mixture are, for certain analyses, dried and purified by resolution in petrol ether. 1 , 2 , 3 The non-lipid extractives, such as urea, glucose, amino acids, and inorganic salts, dissolve in varying amounts in the alcohol-ether, but they do not by themselves dissolve in petrol ether. It has been recognized, however, that the petrol ether solutions show higher N:P ratios than could be expected from any of the known phosphatides. Several attempts have been made to identify the extra nitrogen. 4 , 5 , 6 The present writers have been able to identify most of it as urea, determinable with urease and other urea regeants. Urea by itself is insoluble in petrol ether, but dissolves measurably in it when the blood lipids are present. Measurable amounts of amino acids, determinable by the specific amino acid carboxyl method of Van Slyke and Dillon, 7 are also present. On the other hand, petrol ether fails to redissolve the phosphatides completely. A fraction of them remains in the undissolved residue. It is slight in normal plasmas, but in certain pathological ones it may represent 40% of the phosphatides. It has the following properties suggestive of sphingomyelin: soluble in alcohol, insoluble in petrol ether, N/P ratio of 2, C/P ratio of about 45. All the other lipids seem to be completely redissolved by the petrol ether. Proposed Extraction. The proteins and lipids are precipitated together by colloidal iron, and the water-soluble extractives are washed away. The lipids are then extracted by stirring up the wet precipitate with alcohol and ether.

The Unidentified Base in Gelatin

Van Slyke and Hiller 1 reported evidence that the phosphotungstate precipitate obtained from hydrolyzed gelatin contained amino acid material other than the hexone bases usually found in this precipitate. Other problems prevented continuance of work on the isolation of the material. It was taken up by Van Slyke and Robson 2 but after preparation of a copper salt, since found to be contaminated with proline, the work was again discontinued by Robsons return to England. The unidentified base has now been isolated and recrystallized, both as the picrate and as the hydrochloride, and has the composition of a hydroxylysine. After removal of arginine and histidine as silver salts the residual “lysine fraction” is freed of adherent mono-amino acids by repeated precipitation in very dilute solution as phosphotungstate. The greater part of the lysine is removed by addition of enough picric acid to the hot aqueous solution to combine with 3/4 of the amino nitrogen present. To the mother liquors enough more picric acid is then added to combine with the rest of the amino nitrogen. A picrate of the composition C6H14N2O3. C6H3N3O7 is crystallized, from a dilution of about 1 to 10, and is purified by recrystallization. It is more soluble than the picrate of lysine, and when heated at the rate of a degree every 3 seconds shows a melting point at 225°. Both the nitrogens of the amino acid react completely with nitrous acid in 10 minutes. There is a marked difference from lysine in speed of reaction; in 3 minutes at 25 under the conditions of the manometric analysis 3 lysine evolves only 83% of its total nitrogen, while the new base in the same time evolves 95%.

Gasometric Determination of Carboxyl Groups in Amino Acids

Ruhemann 1 and Grassmann and von Arnim 2 have shown that the color-forming reaction of ninhydrin with amino acids occurs with evolution of CO2. We have found that at pH 5, or somewhat lower, in water solution the CO2 from carboxyl groups adjacent to alpha-amino groups can be quantitatively split off in 3 minutes. The apparatus used is that employed by Van Slyke, Page and Kirk 3 for manometric determination of organic carbon. In the combustion tube of this apparatus are placed a few milligrams of amino acid, 100 mg. of KH2PO4, and 50 mg. of ninhydrin made to a volume of 1.0 cc. with water. The tube is connected to the manometric chamber, arranged as for combustions, 3 and the solution is boiled gently for 3 minutes. The evolved CO 2 is measured as in carbon combustions. 3 The reaction appears to be specific for carboxyl groups of amino acids. Other organic acids, such as acetic, lactic, citric, yield no CO2. Each of the naturally occurring amino acids including proline yields 1 mole of CO2, except the dicarboxylic acids, aspartic and glutamic. Aspartic yields at once 2 moles of CO2, while glutamic yields one mole at once, and a second much more slowly (only about 2% in 3 minutes). Peptides do not react, even with the free carboxyl, which is inactivated by the binding of the alpha NH2 group in peptide linking. Urea does not react. Because other primary amines, urea and ammonia do not interfere, the determination of amino acid carboxyl CO2 may be more specific for amino acids in biological material than is the nitrous acid reaction. Because of the non-reactivity of the free carboxyl group in peptides the method appears to provide a sharp distinction between peptides and free amino acids.

Simplified Colorimetric Determination of Blood Urea Clearance

The urea contents of urine and blood are compared in a colorimeter in such a manner that a single reading gives directly the percentage of average normal renal function in terms of the blood urea clearance. 1 The urine is first diluted to such an extent that if the clearance, either standard or maximum, is the average for a normal subject, the urea concentrations in blood and diluted urine will be equal. The urea in both blood and urine is converted into ammonia with urease; proteins and other interfering substances are removed, and the ammonia contents of the 2 filtrates are compared colorimetrically. No standard solutions are required, because the blood is compared directly with the urine. The number of times the urine must be diluted for comparison with the blood is found by reference to the curve of Fig. 1, computed as follows: The average maximum clearance (for urine volumes over 2 cc. per minute) is 75. The maximum clearance is calculated as Cm = UV/B where V is reckoned in cc. of urine per minute, B is blood urea concentration, and U is urine urea concentration. When C m has the average normal value of 75, the ratio U/B is calculated therefore as U/B = 75/V. When V is expressed in cc. of urine per hour, the calculation changes to U/B = 4500/V. The ratio U/B is the number of times a portion of the urine must be diluted to bring its urea concentration down to that of the blood, when the clearance is average normal. The U/B values thus calculated are expressed by the higher part of the curve of Fig. 1. If the urine volume is below 2 cc. per minute, the formula which holds is that of the standard clearance, Cs = U √ V/B, and the average normal value of Cs is 54.

PHYSIOLOGY OF THE AMINO ACIDS

We have followed the amino acids from their entrance into the alimentary tract in the form of food proteins through the successive steps of digestion, absorption into the blood stream and passage from the blood stream into the tissues, where they are concentrated by some unknown mechanism to many times their concentration in the blood plasma. We have seen something of the way in which certain of the amino acids can be transformed into one another in the body or synthesized from ammonia and keto acids. However, we have had to admit that our bodies can form in such ways only about half of the different amino acids that are required, and that the other half must be made for us by plants, bacteria or other organisms which have greater synthetic powers than we. And finally we have seen something of the manifold fates of the amino acids after they have entered our tissues; how they may be destroyed and their nitrogenous parts turned into urea in the liver before it is possible to put them to their more specialized uses, how their carbon fractions can be used to form glucose, how they may sacrifice themselves to protect us from toxic products, how they can serve as source material for certain vitamins, hormones and other compounds with physiological functions still to be identified, and how finally those amino acids which are not deflected to these various fates may enter into the proteins of the tissues and become for a time parts of our living structures.

Manometric determination of CO2 combined with scintillation counting of C-14☆

Abstract A procedure is described for rapid transfer to scintillation vials of 14CO2 evolved by reactions in the mechanically shaken manometric chamber of Van Slyke and Neill. Results are given with amounts of CO2 ranging from 2 to 1000 μmoles, and with Hyamine and phenylethylamine to absorb CO2 in the vials.

Manometric determination of nitrate and nitrite

Abstract Procedures are described for gasometric determination of nitrate, nitrite, and mixtures of nitrate and nitrite with the Van Slyke-Neill manometric apparatus. Nitrate is measured by the nitric oxide evolved by the reaction with mercury and sulfuric acid: 2HNO 3 + 6Hg + 3H 2 SO 4 = 2NO + 3HgSO 4 + 4H 2 O. Nitrite is measured by the N 2 evolved by reaction with sulfamic acid: HSO 3 · NH 2 + HNO 2 = H 2 SO 4 + N 2 + H 2 O. In mixtures of nitrate and nitrite, the total nitrate plus nitrite nitrogen is determined by adding permanganate to oxidize the nitrite to nitrate and then applying the nitrate method. Nitrite is determined in a separate sample by the reaction with sulfamic acid and nitrate N is calculated as total N minus nitrite N.

Titration of organic acids in urine

Carbonates and phosphates are removed by adding 2 grams of calcium hydroxide to 100 c.c. of urine, and filtering after 10 minutes. 25 c.c. of the filtrate is brought to a pH of approximately 8 by adding 0.2 N HCl with phenolphthalein as indicator, till the pink color disappears. Then 5 c.c. of 0.02 per cent. Tropeolin 00 solution are added, and the solution is titrated to a pH of 2.7 with 0.2 N HCl, the volume being brought to approximately 50 c.c. by addition of water towards the end of the titration. The color is compared with that of 50 c.c. of a control solution with the same pH and indicator. The amount of 0.2 N HCl required to give the endpoint with a control in which water replaces the urine is subtracted. Of the organic acids known to be present in urine in quantitatively significant amounts, the titration measures from 93 to 100 per cent. of each. It also includes very weak bases, but apparently of this class of substances only creatine and creatinine are significant; they are titrated to nearly 100 per cent. The titration figure, corrected for the amounts of these two bases, represents the organic acids.