Soňa Nováková

Analysis of Thiocyanate in Biological Fluids by Capillary Zone Electrophoresis

A new sensitive and simple method has been developed for the determination of thiocyanate in human serum, urine and saliva. The determinations were performed in a fused-silica capillary [64.5 cm (56 cm effective length) x 75 microm] using 0.1 M beta-alanine-HCl (pH 3.50) as a background electrolyte, separation voltage 18 kV (negative polarity), temperature of capillary 25 degrees C and direct detection at 200 nm. Serum samples were 10-times diluted with deionised water and deproteinised with acetonitrile in the ratio 1:2. Urine and saliva samples need only 20-fold dilution with deionised water. The proposed method was successfully applied to the determination of thiocyanate in various human serum, saliva and urine samples.

Inhibition study of angiotensin converting enzyme by capillary electrophoresis after enzymatic reaction at capillary inlet.

Capillary electrophoresis was used to study the inhibition of angiotensin-converting enzyme (ACE) by different inhibitors. Reaction occurred at the capillary inlet during a predetermined waiting period, followed by the electrophoretic separation of the reaction compounds. ACE activity was determined by the quantification of the reaction product, hippuric acid, at 230 nm. The technique was used to study the potency of five different inhibitors (captopril, lisinopril, perindoprilat, quinaprilat and benazeprilat). During a kinetic study, the Ki value of captopril was estimated to be 55.4 +/- 8.8 nM, a value consistent with previously reported values.

Determination of the kinetic parameters of rhodanese by electrophoretically mediated microanalysis in a partially filled capillary.

Electrophoretically mediated microanalysis (EMMA) was applied for the study of kinetic parameters of the bisubstrate enzymatic reaction of rhodanese. The Michaelis constants (Km) for both substrates and the effect of temperature on rhodanese reaction were evaluated by means of the combination of the EMMA methodology with a partial filling technique. In this setup, the part of the capillary is filled with the buffer best for the enzymatic reaction whereas, the rest of the capillary is filled with the background electrolyte optimal for separation of substrates and products. The enzymatic reaction was performed in 25 mM N‐(2‐hydroxymethyl)piperazine‐2’‐(2‐ethanesulfonic acid) (HEPES) buffer (pH 8.5) while the low pH background electrolyte 100 mM β‐alanine‐HCl (pH 3.5) was used for separation of substrates and products that are the inorganic anions. The estimated value of Km for thiosulfate of 1.30×10−2 M was consistent with previously published values; the Km for cyanide of 7.6×10−3 M was determined for the first time. In addition, the type of kinetic mechanism of enzymatic reaction was also elucidated. The finding of the double displacement (ping‐pong) mechanism is in accordance with previous literature data. Also, the experimentally determined temperature optimum of the rhodanese‐catalyzed reaction around 20–25°C agreed with literature values.