Determination of rhodanese enzyme activity by capillary zone electrophoresis.

A new sensitive method has been developed for the determination of rhodanese activity. The enzymatic reactions were carried out directly in thermostatted autosampler vials and the formation of SCN- was monitored by sequential capillary zone electrophoretic runs. The determinations were performed in a 75-micron fused-silica capillary using 0.1 M beta-alanine-HCl (pH 3.50) as a background electrolyte, a separation voltage of 18 kV (negative polarity), a capillary temperature of 25 degrees C and direct detection at 200 nm. Short-end injection or long-end injection procedures were used for sample application. The method is rapid, able to be automated and requires only small amounts of sample and substrates, which is especially important in the case of highly toxic cyanide. The developed capillary electrophoretic method also has great potential for thiocyanate determinations in other applications.

Evidence of complex formation between alkaline phosphatase and a pro-apoptotic hemoprotein cytochrome c.

Two proteins, alkaline phosphatase (AP) and cytochrome c (cyt c) which seem to be involved in the apoptotic cell death program were examined on their interaction. Intestinal AP affects ferricytochrome c (cyt c(FeIII)) by changing its optical properties, redox state and conformation. The effect proceeded over the course of hours with a gradual decrease in free cyt c(FeIII) as the AP concentration increased. A heme containing high molecular species was created in the first stage of interaction of the proteins in neutral, acidic (pH 2.6), alkaline (pH 8.3), low ionic strength (10 mmol/l phosphate), and high ionic strength (0.5 mol/l NaCl) media. Further complexation was favored by higher pH values and temperature. Differential scanning calorimetry revealed a decrease in enthalpy of the thermodenaturation temperature (Tm) of cyt c at 84.5 degrees C due to the AP addition. Increments of AP in the mixtures resulted in the appearance of Tm peaks at 68 degrees C and 61 degrees C. Electrophoretic analysis of the commercial samples of intestinal APs showed main fractions from 63.2 kDa to 72.9 kDa and from 172.9 up to 179.0 kDa. Changes in positions and intensities of the bands were detected upon longer incubation (24 h) with cyt c. The electrophoretic pattern of the bacterial AP was homogeneous with one fraction of 43.7 kDa showing no alteration due to the cyt c presence. Gel permeation chromatography of incubated mixtures of intestinal APs and cyt c confirmed the creation of new heme containing complexes.

Application of capillary zone electrophoresis to study the properties of rhodanese from Acidithiobacillus ferrooxidans.

A new capillary zone electrophoretic method was applied to the assay of enzymic activity of rhodanese from Acidithiobacillus ferroxidans. The enzyme activity determined by capillary zone electrophoresis was compared with that determined by discontinuous spectrophotometry, the values obtained being in good agreement. The method was also used to evaluate Michaelis constants of cyanide and thiocyanate as substrates; a new approach was developed to solve the problem with variable ionic strength of the samples. The pH and temperature optima for the enzyme were also determined.

Formation of iodinin by a strain of Acidithiobacillus ferrooxidans grown on elemental sulfur.

The presence of the pigment iodinin, an Acidithiobacillus ferrooxidans culture metabolite, was demonstrated after growth of bacteria on elemental sulfur. The structure of iodinin was confirmed by X-ray structure analysis; its physiological role is discussed.

Purification and some properties of thiosulfate dehydrogenase from Acidithiobacillus ferrooxidans.

Thiosulfate dehydrogenase was purified from Acidithiobacillus ferrooxidans using three purification steps. The purification procedure involved ammonium sulfate fractionation, ion-exchange chromatography, and gel permeation chromatography. Specific activity of the purified enzyme (after IEC) was 3.26 nkat/mg, and yield of the enzyme was 78%. The purity of the enzyme was checked by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme is a tetramer composed of four probably identical subunits of relative molecular weight 45,000. The pH optimum of the enzyme reaction in the direction of substrate oxidation was found to be 3.0. The isoelectric point of the enzyme was 8.3. Enzyme activity was found to be particularly sensitive to the histidine-selective reagent diethylpyrocarbonate. Reagents selective for arginine, cysteine, and tryptophane had no effect on enzyme activity.

Determination of pyrroloquinoline quinone by capillary zone electrophoresis.

A new method for the determination of pyrroloquinoline quinone by capillary zone electrophoresis has been developed. Separation conditions have been optimised with the respect to different parameters including pH and ionic strength of the background electrolyte, separation voltage and temperature of the capillary. A buffer consisting of 50 mM beta-alanine-HCl pH 3.0 was found to be the most suitable electrolyte for this separation. An applied voltage of 25 kV (negative polarity) and a temperature of 25 degrees C gave the best analysis of pyrroloquinoline quinone. The linear detection range for concentration versus peak area for the assay is from 5 to 500 microM (correlation coefficient 0.9998) with a detection limit of 0.1-0.2 microM. The inter-day reproducibility of the peak area was 2.5% and the inter-day reproducibility of the migration time was below 0.18%.

Purification and properties of malate dehydrogenase from Paracoccus denitrificans.

Affinity chromatography on immobilized Cibacron Blue (Matrex Gel Blue A) gel permeation chromatography on UltroPac TSK G 3000 SWG column and ion-exchange chromatography on "Mono Q" column were used to purify the malate dehydrogenase (MDH) from P. denitrificans to electrophoretic homogeneity. The last two purification steps were performed in FPLC system. The enzyme having a specific activity of about 2300 nkat/mg protein was obtained with an approximate 70% yield. MDH is a dimer with a molecular mass of 80,000 +/- 10,000 and an isoelectric point of 4.85 +/- 0.05. Absorption, fluorescence and CD-spectra were also measured and basic kinetic parameters were obtained for the homogeneous enzyme. The present paper also suggests the possibility of using the prepared enzyme for the determination of aspartate transferase (AST) in blood serum.

Improved chromatographic purification of pea seedlings diamine oxidase.

An improved and simplified purification procedure has been developed for the isolation of diamine oxidase from pea seedlings (DAO EC 1.4.3.6). It involves ammonium sulphate precipitation, hydrophobic interaction chromatography, ion-exchange chromatography and size-exclusion chromatography. The homogeneity of the final enzyme preparations and molecular weight were determined by size-exclusion chromatography and by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS PAGE). The isoelectric point of 7.35 +/- 0.05 was determined by chromatofocusing and by polyacrylamide gel isoelectric focusing.

Detection of quinoproteins after electrophoresis in the presence of urea or SDS.

A method for the direct detection of quinoproteins in gels after electrophoresis in the presence of urea or SDS has been developed. The conditions of sample preparation and detection were optimized to achieve maximum sensitivity. The method is suitable for the detection of quinoproteins in complex protein mixtures as well as for the characterization of certain enzyme preparations.

Use of thiopropyl sepharose for the synthesis of an adsorbent for the affinity chromatography of glutathione S-transferase.

Thiopropyl Sepharose 6B in the 2-thiopyridyl-activated form was used for the reversible immobilisation of reduced glutathione (GSH). The resulting affinity matrix was successfully tested as a sorbent for the partial purification of glutathione S-transferase (GST) from pig kidney. The specific elution of the enzyme was performed with 10 mM GSH in Tris-HCl buffer (pH 7.8), non-specific elution with 20 mM dithiotreitol (DTT) in the same buffer.