Oldřich Janiczek

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.

Metabolic activity of Thiobacillus ferrooxidans on reduced sulfur compounds detected by capillary isotachophoresis

Abstract The sulfur-oxidizing activity of Thiobacillus ferrooxidans was investigated using capillary isotachophoresis. The oxidation of pyrite and sulfur was detected through sulfate formation while oxidation of thiosulfate was detected by thiosulfate consumption. Electrolyte systems and the conditions for the isotachophoretic determination of sulfate (leading electrolyte: HCl, 1 mmol l −1 ; CaCl 2 , 2 mmol l −1 and β -alanine, pH 4.0; terminating electrolyte: citric acid, 1 mmol l −1 ) and thiosulfate (leading electrolyte: HCl, 1 mmol l −1 ; CaCl 2 , 4 mmol l −1 and β -alanine; terminating electrolyte: hexanoic acid, 5 mmol l −1 ) are described. Oxidation of pyrite and sulfur occurred without sulfur intermediate formation; however, during thiosulfate oxidation, sulfur intermediates were detected.

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.

Purification and some properties of thiosulfate dehydrogenase from Acidithiobacillus ferrooxidans.

Abstract 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%.

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.

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

The presence of the pigment iodinin, anAcidithiobacillus 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.

Chromatographic purification of glucose-6-phosphate dehydrogenase and lactate dehydrogenase from Leuconostoc mesenteroides

Abstract A simple procedure for the simultaneous purification of glucose-6-phosphate dehydrogenase and lactate dehydrogenase from Leuconostoc mesenteroides is described. It involves ammonium sulphate precipitation, hydrophobic interaction chromatography and ion-exchange chroamtography. The purity of the final fractions is checked by size-exclusion chromatography and by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The purified enzymes are suitable for enzymological studies and analytical biochemistry.

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 fromAcidithiobacillus ferrooxidans. 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.