Determination Of Amino Nitrogen Research Articles

Comparative analysis of base nutrient composition by NMR spectroscopy

The traditional approach to assessing the quality of nutrient bases involves a determination of amino nitrogen and acidity. The disadvantage of this approach consists in a lack of information, i.e. an inability to detect antibiotics, growth inhibitors and other undesirable compounds. In this regard, more modern and informative methods are required to control the technological process of preparing the nutritional basis and therefore the quality of the products obtained. The aim of this work was to study the physicochemical properties of nutrient bases made from sea and river fish and squid using new approaches (NMR spectroscopy). The following raw materials were used: herring (1), roach (2), pollock (3), squid (4). The raw materials were subjected to enzymatic hydrolysis by the pancreas (according to Hottinger). The qualitative composition of the organic component of hydrolysates (1–4) was determined by 1 H, 13 С and 15 N NMR spectroscopy. All of the 1H NMR spectra had the same appearance, typical of mixtures of amino acids or amino acid sequences. In the high-field part (0.9–2.5 ppm), a set of multiplets was observed, characteristic of aliphatic fragments of molecules. Since most of the signals in the 1H NMR spectra partially overlap, a quantitative assessment of the composition of the organic component appears impossible. All four samples can be confirmed as being qualitatively similar without isolating the dominant compound. Analysis of 2D NMR spectra revealed the presence of the following free amino acids in mixtures of samples (1–4): alanine, valine, threonine, arginine, lysine, leucine, methionine, phenylalanine and glycine. The use of NMR spectroscopy demonstrated that any discrepancies in the component composition of hydrolysates (1–4) were insignificant, allowing manufacturers of nutrient media to choose the most affordable raw materials. The obtained data appear to be applicable for controlling the technological process of preparing the nutrient bases and determining the quality of the resulting products during storage.

The Rhizofiltration of Sodium from Hydroponic Fluid using Scented Geraniums

The ability of two scented geraniums, Pelargonium sp. `Frensham' and Pelargonium sp. `Citrosa' to tolerate and accumulate salt was assessed in a hydroponic system under greenhouse conditions. Rooted cuttings were exposed to a range of salt concentrations (0 to 200 mM NaCl) over a two-week period. No visible signs of phytotoxicity were observed on scented geranium plants grown in solutions up to 100 mM sodium chloride. Tolerance to salt exposure was assessed with chlorophyll a fluorescence parameters. A significant decline in the efficiency of the photosystem (Fv/Fm) was observedin Frensham plants exposed to 200 mM NaCl for 14 days. The numberand size of active reaction centers (Fv/Fo) declined with increasing NaCl treatments in both species. Total chlorophyll content of both species decreased with increasing salt treatmentas a result of significant reductions in the chlorophyll acontent. At higher levels of sodium chloride treatment, salt extrusion was observed on the petioles and the leaf lamina. An accumulation in excess of 3.7 and 2.6% of the dry weight of Frensham plants as sodium was observed in shoots and roots, respectively. The effect of the accumulated sodium on the physiology of scented geraniums was assessed through biochemicalanalysis. The determination of amino nitrogen and stress-relatedmarkers revealed an initial increase in 4-aminobutyrate, proline,asparagine, glutamine, and alanine in the root tissues. Shoot analysis showed gradual increases in asparagine, aspartate, andalanine. These data provide the first evidence for the salt tolerating and accumulating potential of Pelargonium sp. and demonstrate the efficacy of this plant species for the remediation of salt contaminated hydroponic solutions.

Quantitative measurement of endogenous amino acid absorption in unanaesthetized pigs.

The present experiment was carried out with 11 pigs (mean body weight: 53.9 +/- 1.3 kg) fitted with permanent catheters in the portal vein and carotid artery and with an electromagnetic flow probe around the portal vein. They were each subjected to 2 or 3 trials at 3 to 4-day intervals. During each trial the animals received after a previous fasting of 20 h a given amount of a protein-free diet (200 to 1200 g). The blood was collected either continuously for a quantitative determination of amino nitrogen, reducing sugars, urea and ammonia (number of meals 12, mean intake: 727 +/- 60 g) or discontinuously every 30 min between 0 and 8 h after the meal for amino acid analysis (number of meals 8; mean intake 709 +/- 105 g). A rather constant appearance (2 g/h) of amino acids in the portal blood was observed throughout the postprandial period. The intestinal absorption of each amino acid was however variable and represented between 10 and 50% of the daily requirements of the animal during the measuring period (8 h). Glutamine and to a less extent glutamic acid were exceptions as they were taken up by the gut wall from the arterial blood. There was also a marked synthesis of ornithine and citrulline by the latter. Because of the low blood level of urea, there were no apparent exchanges of urea between the blood and the intestine; in contrast, the ammonia absorption represented about 70% of that observed after ingestion of normal protein diets. Most amino acids are largely taken up by the liver and peripheral tissues, but in the case of alanine the syntheses exceed the uptake.

The effect of proteolytic enzymes on the thermostability of egg-white.

The effect of proteolytic enzyme preparations such as Proteopol PB (bacterial origin), ficin, and pancreatin on the thermostability of egg-white proteins during pasteurization was investigated. Proteolytic activities of each enzyme were measured at three different doses which were calculated by the ANSON method. The degree of white protein degradation was evaluated by the determination of amino-nitrogen. Effectiveness in reducing the coagulation of egg-white proteins varied, depending on the type and dose of enzyme used. Proteopol PB showed only little effect, ficin demonstrated the best anticoagulation properties at the lowest dose, while for pancreatin the middle dose proved to be the most effective. It was concluded that moderate proteolysis is useful for improving the thermostability of egg-white proteins during pasteurization. Total bacterial counts increased considerably after incubation of egg-white with enzyme preparations, but after pasteurization the number of bacteria decreased to levels which were compatible with bacteriological standards for egg-white products.

A PROCEDURE FOR SCREENING FOR PROTEIN CONTENT IN CEREAL BREEDING

A method for the estimation of grain protein content based on the automated determination of amino nitrogen in protease digests of grain was found to be a possible alternative to the Kjeldahl procedure.

Implication of an Ionizing Group in the Control of Conformation and Activity of Chymotrypsin

Abstract 1. Acetylated chymotrypsinogen was prepared by the reaction of crystallized chymotrypsinogen with acetic anhydride at pH 6.7 and 4°, and purification of the product. End group analysis and Van Slyke amino nitrogen determination indicated that all the free e- and α-amino groups of the chymotrypsinogen molecule had been blocked by acetylation. 2. Activation of acetylated chymotrypsinogen by trypsin, followed by chromatography, yielded acetylated δ-chymotrypsin. Quantitative end group determination indicated that the molecule had a single amino group, the NH2-terminal α-amino group of an isoleucine residue. In the catalytic hydrolysis of N-acetyl-l-tryptophan amide, the steady state kinetic parameters were found to be the same, in the pH region 6 to 10, with acetylated δ-chymotrypsin and with δ-chymotrypsin. 3. The activation of acetylated chymotrypsinogen to acetylated δ-chymotrypsin was found to be accompanied by the appearance of a single new ionizing group, titratable in the pH region 5 to 10.5, with a pK(app) of 8.3. 4. The specific rotation of acetylated δ-chymotrypsin was found to vary with pH in the pH region 6 to 10.5, the change in specific rotation following the ionization of a single group with pK(app) of 8.3. Above pH 10 the specific rotation of the zymogen and the enzyme were found to be the same at the three different wave lengths investigated. 5. Previous investigations in this and other laboratories of the pH dependence of the quotient of the steady state kinetic parameters, kcat [Km(app)]-1 —a function which yields the ionization constants of the catalytically importance ionizing groups of the free enzyme—have indicated the importance of an ionizing group with pK(app) ∼ 8.5 in all α-chymotrypsin-catalyzed reactions studied, and in the δ- or acetylated δ-chymotrypsin-catalyzed hydrolysis of N-acetyl-l-tryptophan amide. 6. An interpretation of these results that is consistent with all available experimental information is that the conversion of catalytically inactive chymotrypsinogen to active enzyme is accompanied by the appearance of a new ionizing group (a group that has a pK(app) of 8.3 in acetylated δ-chymotrypsin) which controls the conformation and thereby the activity of the enzyme. Molecules in which this ionizing group is unprotonated are in a catalytically inactive conformation, resembling that of chymotrypsinogen, and molecules in which this ionizing group is protonated are in a catalytically active conformation. A diisopropyl fluorophosphate (DFP)-induced protein isomerization involves only the enzyme molecules that are in the catalytically active conformation. This interpretation leads to a prediction that in the presence of DFP there will be an increase in pK(app) of the ionizing group. Such an increase in pK(app) was previously observed, and accounts for the proton uptake and the change in specific rotation of the enzyme observed in its reaction with DFP. 7. All evidence obtained so far suggests that the group with pK(app) ∼8.5 is an NH2-terminal isoleucyl α-amino group.

Changes in Amino-nitrogen Content of Solutions of γ-Keratose from Wool Keratin

NUMEROUS workers1–3 have fractionated wool keratin after oxidation of disulphide bonds with performic or peracetic acids into three fractions, α, β and γ-keratose (β-keratose is insoluble in water, α-keratose is that fraction precipitating at pH 4 and γ-keratose the remaining fraction). While these fractions vary somewhat in the amounts of terminal amino-acids4, no comments have yet been made on any changes in constitution of the isolated fraction of γ-keratose on re-dissolution in water. As an introduction to some work on the hydrolysis of γ-keratose (prepared by the method of Corfield, Robson and Skinner2), the free amino-nitrogen value was determined by reaction with nitrous acid, the method of Van Slyke5,6, and it was found that reproducible results were not obtained on samples until the solution had been standing for some time. Accordingly the determination of amino-nitrogen of γ-keratose (10 mg/ml.) at various periods of time after dissolution was carried out and plotted as a function of time in acid (0.2 M potassium hydrogen phthalate buffer, pH 4), alkaline (0.2 M boric acid in 0.2 M potassium chloride buffer, pH 10), salt (0.2 M potassium chloride) and simple aqueous solution. The results are shown in Fig. 1.

Dimethyl sulfoxide as solvent for amino nitrogen determination on cereal proteins by the Van Slyke manometric method

Dimethyl sulfoxide as solvent for amino nitrogen determination on cereal proteins by the Van Slyke manometric method

Determination of amino nitrogen, pyrrolidone carboxylic acid nitrogen, and total nitrogen with ninhydrin

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Zur Konstitution von Hypoglycin B

AbstractN‐terminal amino acid analysis, VAN SLYKE amino nitrogen and VAN SLYKE carboxyl determinations give evidence that hypoglycin B is γ‐L‐glutamyl‐hypoglycin‐A. VAN SLYKE amino nitrogen determinations by the method of G. KAINZ et al. demonstrate that it is possible, at least in the case of hypoglycin B, to avoid the anomalous behavior (reaction of both the amino and the γ‐amide groups with nitrous acid) of γ‐glutamyl peptides with a free α‐carboxyl group.

Colorimetric determination of amino nitrogen in colored solutions

Colorimetric determination of amino nitrogen in colored solutions

Studies in cystic fibrosis; determination of plasma proline following protein feeding as a diagnostic test for pancreatic insufficiency.

An earlier study1suggested a diagnostic procedure for pancreatic insufficiency associated with cystic fibrosis of the pancreas based upon the determination of plasma glycine following gelatin feeding. West, Wilson, and Eyles2had shown that a much smaller rise in blood amino nitrogen occurs in children with cystic fibrosis than in normal children after a test meal of casein. Christensen and Shwachman1confirmed the suggestion that gelatin might serve as a more suitable test protein and were able to avoid (1) the technical difficulties involved in the manometric ninhydrin determination of amino nitrogen or (2) the use of unsatisfactory colorimetric amino nitrogen methods by determining the change in plasma glycine concentration by a suitable method.3The selection of a single amino acid for estimation was based on the fact that gelatin contains somewhat more than 25% of glycine. The method is, however, not suitable for diagnostic purposes

Electrophoretic behaviour in filter paper and molecular weight of insulin

Insulin travels as well defined band in electropherograms if acetic acid-water 1:2 (v/v) is used as a buffer. Preparations of partially acetylated insulin were analysed by this method. From the results it could be derived that the molecular weight of insulin is 6,000. An improvement in the amino-nitrogen determination of insulin is described in the experimental part.

Effect of beef spleen extract on the respiration of normal mouse tissues during storage.

SummaryBy means of Warburg respirometers the oxygen consumption of brain, connective tissue, heart, kidney, liver and lung from normal dba mice was measured at 48-hour intervals during a period of 18 days of storage at 4°C, in Tyrode's solution and in beef spleen extract. The respiration of all six of the normal tissues, whether stored in Tyrode's solution or in beef spleen extract, decreased during the initial 8- to 10-day period, while the same tissues, with the exception of liver, were characterized by a progressive increase in respiration during the 12- to 18-day period of storage. This increase could not be correlated with bacterial contamination or with tissue autolysis as revealed by histologic observation and amino nitrogen determination. The immediate effect of the extract was to decrease the oxygen consumption of brain, lung and heart, and, to a lower degree, of liver and connective tissue, while that of kidney was increased. The effect of beef spleen extract on dbrB adenocarcinoma in dba mice f...

Aminoaciduria in Progressive Muscular Dystrophy.

Two methods, partition chromatography and chemical amino nitrogen determination, have been employed in a study of the amino-acid excretions of 32 patients diagnosed as suffering from progressive muscular dystrophy and closely related conditions. In the case of progressive muscular dystrophy, partition chromatography indicated that both the number and the concentration of amino acids excreted were in many cases considerably above the normal limits. In addition, the results of amino nitrogen determinations indicated that some of these patients showed values far above normal limits and the majority were somewhat greater than normal.

Determination of Amino Nitrogen in Corn Sirup

Determination of Amino Nitrogen in Corn Sirup

Le globucid (n. d.) essais d' identité et dosages

This analytical study of Globucid (sulphanilaminoethylthiodiazole) comprises : l. The determination of physical constants, melting-point and solubilities, of the compound. 2. The identification of the different functional groups, arylamine, substituted benzene, sulphonamide and tests for aminoethylthiodiazole by colour and precipitation reactions, as well as a number of precipitation and microchemical reactions of Globucid itself. 3. A quantitative section : determination of total nitrogen and thiodiazole nitrogen by difference, determination of amino nitrogen, acidimetric and argentometric determination of the sulphonamide groups, determination of total sulphur and thiodiazole sulphur and finally bromometric assay of Globucid.

Penicillamine, a Characteristic Degradation Product of Penicillin

WE noticed more than nine months ago that acid hydrolysates of penicillin gave a strong blue-violet coloration with the ninhydrin reagent. Afterwards it was found that, under standardized conditions of hydrolysis, the colour intensity given by numerous penicillin preparations of varying degrees of purity ran parallel to their antibacterial activities except in the case of very crude specimens. It was also found that about 59 per cent of the total nitrogen of the purest preparations could be estimated as amino-nitrogen after one hour's hydrolysis by means of N/10 sulphuric acid under the conditions of the van Slyke method. These facts indicated that the substance responsible for the ninhydrin reaction and the positive results in the van Slyke amino-nitrogen determinations was a fundamental part of the penicillin molecule. Its isolation was therefore regarded as a matter of importance and we are now able to report that it has been obtained as a crystalline hydrochloride. The properties of this substance, which we propose to term penicillamine, show that it represents a novel type of naturally occurring base. Penicillamine is identical with the base which we have stated in an earlier communication1 to occur in acid penicillin hydrolysates.

SEROLOGICAL REACTIVITY OF HYDROLYTIC PRODUCTS FROM SILK

The foregoing experiments show that products of the hydrolysis of silk that consist of peptides having, from amino nitrogen determinations, molecular weights from about 600 to 1000 were capable of inhibiting the reactions of precipitin sera for silk. From the results it may reasonably be inferred that silk fibroin contains determinant structures not larger than the peptides examined, which probably consist of not more than 8 to 12 amino acids. That similar relations may obtain with other proteins is not improbable in view of results previously reported with dialyzable split products.

CONSTITUENTS OF ELEMENTARY BODIES OF VACCINIA

The effects of a number of crystalline and highly purified enzymes on elementary bodies of vaccinia are reported. These effects have been followed by determination of amino nitrogen, staining reaction, and studies of infectivity. Pepsin, at a pH which inactivates the virus, results in its solution and rapid release of amino nitrogen. Crystalline trypsin, chymotrypsin, carboxypeptidase, and ribonuclease are without appreciable effect on the virus. Papain within a short time produces profound alteration in the staining reaction of the elementary body with release of amino nitrogen accompanied by complete inactivation of the virus. This reaction is not shared by crystalline ficin, another plant papain, or by cathepsin, an intracellular proteinase analogous to plant papains but of animal origin.