Biomedical subjects

Sona Nováková

Publications and source records attributed to Sona Nováková.

6 recordsLinked to original sources

PubMed2004

Study of enzyme kinetics of phenol sulfotransferase by electrophoretically mediated microanalysis.

Electrophoretically mediated microanalysis (EMMA) was applied for the study of the kinetic parameters of the enzymatic reaction of phenol sulfotransferase SULT1A1 isoenzyme with 4-nitrophenol as a substrate. The SULT1A1 activity was determined by the quantitation of the product, 4-nitrophenyl sulfate, at 274 nm by using different injection and separation steps. This new approach solved the problem of the presence of the very strong inhibitor, adenosine 3',5'-bisphosphate (PAP), in the co-substrate solution (adenosine 3'-phosphate 5'-phosphosulfate, PAPS) which is unstable at room temperature. The inhibitor PAP was electrophoretically separated from the co-substrate PAPS before the injection of enzyme and substrate inside the capillary (and thus before their in-capillary encountering). With the developed in-capillary SULT1A1 activity assay an average Michaelis constant (Km) for 4-nitrophenol was calculated to be 0.84 microM, a value which is consistent with a previously reported value. Strong substrate inhibition (above a 4-nitrophenol concentration of 2.5 microM) was observed, and this is also in accordance with literature values.

Adenosine Diphosphate↗

PubMed2004

Electrophoretically mediated microanalysis.

This review describes the existing developments in the use of the capillary electrophoretic microanalytical technique for the in-line study of enzyme reaction, electrophoretically mediated microanalysis (EMMA). The article is divided into a number of parts. After an introduction, the different modes, basic principle, procedure, and some mathematical treatments of EMMA methodology are discussed and illustrated. The applications of EMMA for enzyme assay and for non-enzymatic determination are summarized into two tables. In addition to classical capillary electrophoresis (CE) instrument EMMA, special emphasis is given to a relatively new technique: EMMA on CE microchip. Finally, conclusions are drawn.

Electrophoresis, Capillary↗

PubMed2003

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.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed2003

Inhibition study of rhodanese by means of electrophoretically mediated microanalysis.

A combination of the electrophoretically mediated microanalysis methodology with a partial filling technique was applied for the inhibition study of bovine liver rhodanese by 2-oxoglutarate. In this set-up, part of the capillary is filled with the best buffer for the enzymatic reaction, while the rest of the capillary is filled with the optimal background electrolyte for separation of substrates and products. The estimated value of K1 for 2-oxoglutarate was 3.62 x 10(-4) +/- 1.43 x 10(-4) M with respect to cyanide and 1.40 x 10(-3) +/- 1.60 x 10(-4) M with respect to thiosulfate. In addition, the type of inhibition was also evaluated. The findings of 2-oxoglutarate as the competitive inhibitor with respect to cyanide and as the uncompetitive inhibitor with respect to thiosulfate are in accordance with previous literature data.

Electrophoresis↗

PubMed2003

Advances in capillary electrophoretically mediated microanalysis.

Recent developments in the use of capillary electrophoretic techniques for the in-line study of enzyme reactions and derivatization protocols are reviewed. The article is divided into two parts: (i) in-line enzyme reactions and (ii) in-line derivatization. The first part introduces electrophoretically mediated microanalysis (EMMA) and discusses and illustrates the different modes of EMMA. A literature overview is provided, starting from 1996, and the investigated enzymes are classified into two tables based on the mode of engagement (i.e., continuous or transient) of the developed EMMA-based assay. The second part starts with an introduction of the procedures and the nomenclature used in the area of in-line derivatization protocols based on EMMA. Reported derivatization procedures are discussed and classified in tables, according to the functional group that is derivatized.

Electrophoresis, Capillary↗

PubMed2002

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 (K(m)) 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 beta-alanine-HCl (pH 3.5) was used for separation of substrates and products that are the inorganic anions. The estimated value of K(m) for thiosulfate of 1.30 x 10(-2) M was consistent with previously published values; the K(m) for cyanide of 7.6 x 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 degrees C agreed with literature values.

Electrophoresis, Capillary↗