A method for the quantitative determination of hypoglycin A in the presence of leucine.

A spectrophotometric method for the quantitative determination of the toxic amino acid hypoglycin A in mixtures with leucine is reported. Hypoglycin A is the hypoglycemic principle found in the arilli of the fruit of Blighia sapida (ackee).     

A rapid method for the determination of cellular protein in the presence of elemental sulfur.

Cellular protein in the presence of elemental sulfur is determined by the Folin reagent after treatment with benzene. Using this procedure a generation time of 46.2 +/- 4.2 h was observed for Thiobacillus acidophilus grown on elemental sulfur.     

A modified colorimetric method for the determination of orthophosphate in the presence of high ATP concentrations.

We describe a modified colorimetric method that quantitates inorganic phosphate linearly up to 60 nmol, with high stability of the developed color and with a low interference by ATP concentration (up to 30 mM). This method is very suitable for use in ATPase enzymatic assays, especially with enzymes that have low specific activities and (or) high Km values for ATP.     

A spectrophotometric method for determination of bacterial biomass in the presence of a polymer

A simple and rapid spectrophotometric method based on a single optical density measurement was developed to quantify biomass in the presence of a growth - associated exopolymer. It allowed the determination of both cell and polymer concentrations in a cultivation broth without the need for separation of these two elements. Cultivations of Pseudomonas fluorescens NCIMB 11671 in a fermenter were taken as examples to demonstrate the validity of this approach. © Rapid Science Ltd. 1998     

A spectrophotometric method for the determination of gamma-glutamyl cyclotransferase with alanine dehydrogenase in the presence of anthglutin

gamma-Glutamyl cyclotransferase activity is assayed in tissues by a colorimetric method using gamma-glutamyl alanine as a substrate coupled with alanine dehydrogenase from B. sphericus, to measure the formation of NADH. In order to avoid interference by the reaction catalyzed by gamma-glutamyl transpeptidase, anthglutin, a specific inhibitor of the transpeptidase was included in the reaction mixture. The Km value of rat kidney gamma-glutamyl cyclotransferase with respect to gamma-glutamyl alanine appeared to be the same when determined by either the colorimetric or the radiometric method. This assay presents a reliable alternative to the use of radiolabeled substrate and is used for the assay of gamma-glutamyl cyclotransferase in a variety of physiological and experimental samples.     

A method for the determination of hyaluronate in the presence of other glycosaminoglycans and its application to human intervertebral disc.

A highly sensitive proteoglycan-binding assay was developed for the detection and determination of small amounts of hyaluronate. Application of this assay to human intervertebral-disc tissue showed that the radial distribution of hyaluronate closely followed the distribution of proteoglycans.     

pH effects in plasmin-catalysed hydrolysis of alpha-N-benzoyl-L-arginine compounds.

The steady-state kinetics of plasmin (EC 3.4.21.7) catalysed reactions with some alpha-N-benzoyl-L-arginine compounds is investigated in the pH range 5.8--9.0. The results are interpreted in terms of a three-step mechanism, which involves enzyme-substrate complex formation, followed by acylation and deacylation of the enzyme. Alpha-N-Benzoyl-L-arginine methyl ester and ethyl ester show the same pH behaviour. The kinetic parameter kc/Km is influenced by two groups with pK values of 6.5 and 8.4, respectively. kc is affected only by the group with pK equal to 6.5 and Km only by the group with pK equal to 8.4. It is suggested that the group with pK equal to 6.5 is the 1-chloro-3-tosyl-amido-7-amino-2-heptanone-sensitive histidine residue in the active site and that the group with pK equal to 8.4 is perhaps the alpha-amino group of the N-terminus in analogy to trypsin and chymotrypsin. alpha-N-Benzoyl-L-arginine amide is not hydrolysed by plasmin, but proves to be a competitive inhibitor, Ki = 12.8 +/- 1.8 mM, pH = 7.8. Also the product alpha-N-benzoyl-L-arginine is a competitive inhibitor, Ki = 26 +/- 3.1 mM, pH = 7.8. Estimates of individual rate constants are compared with similar trypsin data.     

Studies on the heterotropic interaction of hemoglobin. I. Mass spectrometric method for determination of the pKa of the beta-146 histidine residue in human hemoglobin.

A mass spectrometric method was developed to determine pH-dependent hydrogen-deuterium exchange at the C-2 position of the imidazole ring of histidine, after converting the amino acid to the methylthiohydantoin derivative. The amount of deuterium exchange in N-acetyl-histidine estimated by the present method was confirmed to be in good agreement with that determined by NMR spectrometry. N-Acetylhistidine was deuterated at various pH's. From the amount of deuterium exchange, a pseudo-first order rate constant (kpsi) was calculated. A pKa value of 7.2 for the amino acid was obtained from the relation between kpsi and pH. This method was applied to estimate the pKa value of beta-146 histidine in human hemoglobin. Human hemoglobin deuterated at various pH's was digested with carboxypeptidase A [EC 3.4.12.2] to release the beta-146 histidine. The amount of deuterium exchange in the isolated histidine was determined to obtain kpsi. From these measurements pKa values of 7.0 for the histidine in oxyhemoglobin and of 8.2 for that in deoxyhemoglobin were found at 36.5 degrees, respectively.     

A new method for the determination of microbial activity and critical logP in the presence of organic solvents

A new method to determine microbial activity and critical logP of an organism in the presence of organic solvents has been developed which involves direct contact with a solvent, and a measurement of the developing colony size. This technique has been used to estimate the critical logP of Alcaligenes xylosoxidans Y234, and, although the critical logP for this organism is 3.5, solvents with logP values of up to 4.5 can still reduce microbial activity by up to 55% of the uninhibited amount.     

A spectrophotometric method for the direct determination of cysteine in the presence of other naturally occurring amino acids

1. An acid ninhydrin reagent was found to react specifically in forming a pink product (E(max.) 560mmu) with cysteine. 2. The method was highly sensitive for the determination of cysteine (in 28.0x10(3)). Homocysteine, glutathione, proline, ornithine and other naturally occurring amino acids tested did not give a similar reaction. 3. The reaction product was stable for at least 3-4hr. at room temperature and the extinction was proportional to the concentration in the range 0.05-0.5mumole of cysteine. 4. The acid ninhydrin reagent also gave yellow products (E(max.) 370-404mmu) with tryptophan, 5-hydroxytryptophan, 5-hydroxytryptamine and indol-3-ylacetic acid. 5. The method was applied for the determination of cysteine in perchloric acid extracts of rat brain, liver and blood.     

A method for estimating phosphate in the presence of phytate and its application to the determination of phytase

Quantification of coenzymes and related compounds from methanogens was performed in extracts obtained from whole cells with aqueous ethanol at 80°C. By means of high-performance liquid chromatography the following compounds could be detected and quantified in extracts from Methanobacterium thermoautotrophicum: coenzyme MF₄₃₀, the prosthetic group of methylcoenzyme M reductase, F₅₆₀, an oxidation product of this compound, coenzyme F₄₂₀, F₃₄₂, methanopterin, and carboxytetrahydromethanopterin, previously known as YFC. Coenzyme MF₄₃₀, coenzyme F₄₂₀, and methanopterin could be determined in extracts from Methanosarcina barkeri. Structural differences were noticed between the coenzymes from the methanogenic bacteria studied.     

A spectrophotometric method for determination of sphingomyelinase.

A colored derivative of sphingomyelin was synthesized and used as substrate for several sphingomyelinases. The compound is N-omega-trinitrophenyl-aminolaurylsphingosylphosphorylcholine. The rate of hydrolysis of this substrate was compared to that of bovine brain sphingomyelin, labelled with tritium in the choline moiety. The following enzyme preparations were used: homogenate-less debris of brain, assayed at pH 5.0 or 7.4; a solubilized preparation derived from rat brain lysosomes, assayed at pH 5.0 and a purified enzyme of Staphylococcus aureus. With all preparations, the rates of hydrolysis of the yellow derivative were very similar to those of the brain sphingomyelin. Extracts of skin fibroblasts of normal and Niemann-Pick patients as well as amniotic cells were also used. Again, the rates of hydrolysis of the yellow derivative practically equalled those using brain sphingomyelin.