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A kinetic study of ornithine carbamyl transferase (OCT) from Streptococcus faecalis has been carried out, employing a new HPLC method based on the direct determination of citrulline. A different kinetic pattern was observed in tris(hydroxymethyl)-aminomethane (TRIS) and triethanolamine (TEA) buffers: a ping-pong or an ordered sequential mechanism are suggested, respectively. The inhibition of Tris buffer on OCT reaction has also been demonstrated. The inhibition of Tris buffer on OCT reaction has also been demonstrated.

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A study of complex formation equilibria of some beta-amino-alcohols with lead(II) and cadmium(II) ions at 25 degrees C and in 0.5 M KNO(3) is reported. The amino-alcohols considered are 2-amino-1-propanol, 2-amino-1-butanol, 2-amino-1-pentanol and 2-amino-1,3-propanediol. sec-Buthylamine and 2-amino-1-methoxy-propane have been also considered for comparison. The results are discussed in terms of ligand structure, paying attention to the number of hydroxyl groups and to the length of the alkyl residual. A weak contribution of the alcoholic oxygen in the coordination of cadmium(II) and the presence of a mixed hydroxyl species in lead(II) containing systems are hypothesized.

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A simple high-performance liquid chromatography (HPLC) assay for the simultaneous determination of guanase and aspartate aminotransferase (AST) activities in a single serum sample is described. The method is based on direct detection of enzymatically formed products xanthine and glutamate, respectively. The procedure is sensitive, precise (C.V. below 2% for guanase and 3% for AST), suitable for routine purposes and requires only 100 microliters of sample. Kinetic measurements have shown the guanase activity to have an apparent Michaelis constant of 24.5 microM and the AST activity of 11.1 and 0.18 mM for aspartate and oxoglutarate, respectively, at 37 degrees C in Tris-HCl buffer (pH 7.5).

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Formation constants of the silver(I) complexes with some amino-alcohols have been determined at 25 degrees C in 0.5 M KNO(3) by means of two independent potentiometric measurements employing glass and silver electrode. The ligands considered are: sec-butylamine, 2-amino-1-propanol, 2-amino-1-methoxy-propane, 2-amino-2-methyl-1-propanol, 2-amino-1-butanol, 2-amino-1-pentanol, 2-amino-1-hexanol, 2-amino-1,3-propanediol, 2-amino-1,3-hexandiol, 2-amino-2-methyl-1,3-propanediol and Tris(hydroxymethyl)-aminomethane. Protonation constants of the selected ligands have also been determined. Calculations were made using HYPERQUAD computer program. The influence exerted by the introduction of hydroxy groups and by the presence of alkyl residuals in the ligand structure on the formation equilibria, is discussed.

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A new assay for argininosuccinate lyase based on the separation of the enzymatic reaction mixture components by an ion-pair reversed phase mechanism is reported. The determination of enzyme activity is performed after direct injection by UV detection of the fumarate formed. The chromatographic analysis time is 8 min and a detection limit of 0.4 U/L is achieved. The HPLC method is highly accurate, sensitive and precise. The simple procedure makes this method suitable for the routine determination of ASAL activity in human serum samples.

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A sensitive and reproducible assay of aspartate aminotransferase activity based on UV detection of the reaction products after their separation by HPLC is described. The main advantage is the direct measurement of the enzyme activity as micromoles of product (glutamate) formed within a known period of time without any coupled reaction. Further, with the chromatographic method, all components of the reaction mixture are identified, allowing the reaction course to be controlled and the possible presence of side-reactions to be monitored.

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A rapid isocratic high-performance liquid chromatographic method for the determination of guanase (EC 3.5.4.3) activity is proposed. The method is highly reproducible, with a coefficient of variation of less than 1%, and requires only ca. 10 min for a complete chromatographic separation of the enzyme reaction mixture. The method allows the detection of nanomolar changes in the concentrations of both the substrate and the product, and does not require additional reactions or sample pretreatment. Kinetic studies with the proposed method showed the guanase activity to have an apparent Michaelis constant of 13.3 and 8.5 microM, and a maximum rate of 1.95 and 3.84 pmol/min per mg protein at 37 degrees C, in Tris-HCl and phosphate buffer, respectively.

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Thermodynamic characteristics pertinent to the formation equilibria of two ternary systems: 1) Copper(II), 4,9-diazadodecane-1,12-diamine (spermine, Spe), and adenosine 5'-triphosphate (ATP) and 2) Copper(II), Spe, and tripolyphosphate (TPP) have been determined by means of potentiometric and calorimetric techniques, together with the parent binary complex characteristics. Ternary complexes involving ATP can give information useful in the interpretation of bioenergetic reactions and of biological interactions between nucleic acids and polyamines. As a model system, the TPP-containing ternary complexes have been studied, together with the parent binary complexes. The thermodynamic study of these systems is very important because it can give information about the structural environment of the complexes; moreover, it can help in outlining different noncovalent interactions such as coulombic forces and hydrogen bonds.

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Thermodynamic parameters pertinent to the equilibria of ternary system copper(II), adenosine 5'-monophosphate (AMP), and 4,9-diazadodecane-1,12-diamine (Spermine, Spe) were determined by means of potentiometric and calorimetric techniques, together with the corresponding parent binary complex parameters. This system could be considered as a model in the study of more complicated biological systems. In particular, it could give useful evidence in the investigation of the interaction between tetramine and DNA. The experimental results seem to suggest that the major contribution in DNA stabilization by means of tetramines is due to the electrostatic interactions occurring between the protonated nitrogen atoms of the amine molecules and the phosphate groups of nucleotides in the nucleic chains.

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A rapid, simple method for resolution and detection of mixtures of aliphatic aldehyde by thin-layer chromatography of their MBTH derivatives is proposed. Less than 1 mg l . levels of each aldehyde in the original aqueous solution can be detected.

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The values of conditional formation constants for the cobalt(II) chloride-1-nitroso-2-naphthol system were determined at 25 degrees in ethanol-benzene mixtures of different compositions by spectrophotometry in the visible region. Comparisons were made with the results previously obtained with the isomeric ligand 2-nitroso-1-naphthol. In both cases the L/M complexation ratio of the species found increases with increasing ethanol content of the medium.

RESUMEN

The cobalt(II) chloride-2-nitroso-1-naphthol system has been studied spectrophotometrically in a 96:4 (v v ) benzene-ethanol solution. Two species have been found, the M:L complexation ratios of which are 1:1 and 1:2. The concentration quotients of the formation equilibria are K(1) = 1.2 x 10(3) and K(2) = 1.4 x 10(4) respectively. The first step of the reaction is accompanied by a configurational change from a tetrahedral to an octahedral structure.

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