[A spectrophotometric method for determining the concentration of L-lysine using L-lysine-alpha-oxidase from Trichoderma sp. and 3,3',5,5'-tetramethylbenzidene dihydrochloride]. / Spektrofotometricheskii metod opredeleniia kontsentratsii L-lizina s pomoshch'iu L-lizin-alpha-oksidazy iz Trichoderma sp. i 3,3',5,5'-tetrametilbenzidindigidrokhlorida.

A simple and relatively sensitive procedure was developed for determination of L-lysine at 3-30 mmole/L concentration. The procedure does not involve the carcinogenic compound o-dianisidine. L-lysine alpha-oxidase catalyzed oxidative deamination of L-lysine with O2 consumption and formation of H2O2, NH3 and alpha-keto-epsilon-aminocaproic acid. Horseradish peroxidase and a non-carcinogenic compound 3,3,5,5'-tetramethylbenzidine dihydrochloride as an electron donor were used in determination of H2O2 formed. The procedure developed enabled also to measure the L-lysine alpha-oxidase activity at the enzyme concentrations of 10-500 ng/ml. The only limitation of the procedure is relatively low pH-values of the reaction medium.

Determination of the catalytic activity of cyclomaltodextrin glucanotransferase by maltotriose-methylorange assay.

A kinetic assay of cyclomaltodextrin glucanotransferase (EC 2.4.1.19), based on the use of methylorange dye as the indicator of cyclodextrins, was studied and the reaction verified with independent HPLC analyses. The assay was optimized for the enzyme of an alkalophilic Bacillus sp. (ATCC 21783). Described enzymological data provide a reasonable background for detection of possible errors. Numerical correlations with previous assays are presented. This method can be adopted as a standard analysis of any cyclomaltodextrin-forming enzyme.

[31P-NMR spectra of aspartate aminotransferase from cytosol of the chicken heart]. / Issledovanie spektrov 31P-IaMR aspartat-aminotransferazy iz tsitozolia kurinykh serdets.

31P NMR spectra of the cytosolic chicken aspartate aminotransferase have been recorded at 161.7 MHz in the pH range of 5.7 to 8.2. The 31P chemical shift was found to be pH-dependent with a pK of 6.85; difference in the chemical shift at pH 5.7 and 8.2 is only 0.35 ppm. The monoanion-dianion transition of 5'-phosphate group of a model Schiff base of pyridoxal phosphate with 2-aminobutanol in methanol is accompanied by a change in 31P chemical shift of 5.2 ppm. It is inferred that the phosphate group of the protein--bound coenzyme is in dianionic form throughout the investigated pH range; the small pH-dependent change of chemical shift may be due to a protein conformational change that affects O-P-O bond angle. In the presence of the 0.1 M succinate, 31P chemical shift of the enzyme remains constant in the pH range of 5.0 to 8.3.

Affinity chromatography of B6-vitamin-dependent enzymes: purification of pig-heart branched-chain amino acid transaminase.

Pig-heart branched-chain amino acid transaminase (EC 2.6.1.42) was purified to near homogeneity with a yield of 27%. A prepurification was performed by heat treatment, gel chromatography and DEAE-Sepharose methods. For the final step, several affinity gels were tested and the one containing cycloserine coupled to CNBr-activated Sepharose 4B was selected. This effected an additional five-fold purification with a yield of 60%. The present affinity results are compared with corresponding studies with other aminotransferases in an attempt to find possible universal techniques for their purification.

A specific arginase-based assay for L-canavanine in leguminous plants.

A convenient method for the analysis of free L-canavanine in leguminous plants is described. Canavanine was specifically hydrolyzed to canaline and urea by the enzyme arginase (EC 3.5.3.1). The resulting amino-oxy functions of canaline were measured based on their ability to bleach the yellow colour of pyridoxal 5'-phosphate. Canavanine in the seeds of Canavalia ensiformis was determined with this method.

Temperature effects in affinity chromatography of alanine aminotransferase.

The effects of temperature on the elution parameters of alanine aminotransferase and albumin were studied on substituted agaroses designated for the affinity chromatography of the enzyme. The elution volume of alanine aminotransferase depended logarithmically and and the elution volume of albumin linearly on temperature. Both decreased when the temperature increased. It was concluded that the observed elution volume of alanine aminotransferase was due to two types of retardation mechanisms: specific (the logarithmic mode) and non-specific (the linear mode), both of which were additive. Thermodynamic parameters were estimated for the specific mode and the calculation resulted in delta Ho and delta So values of ca. -40 kJ/mole and -140 J/mole . degrees K, respectively.