Study of Enzymatic Reaction by Electrophoretically Mediated Microanalysis in a Partially Filled Capillary and Indirect or Direct Detection
Telnarova, M., Vytiskova, S., Chaloupkova, R., Glatz, Z.
ELECTROPHORESIS 25: 290-296 (2004)
This report describes the application of electrophoretically mediated microanalysis (EMMA) in combination with a partial filling technique and indirect or direct detection for the study of enzymes dealing with the high mobile inorganic or organic anions as substrates or products. In this set-up the part of the capillary is filled with the buffer best for the enzymatic reaction whereas the rest of the capillary with the background electrolyte optimal for separation of substrates and products. In the case of haloalkane dehalogenase, which was chosen as a model enzyme, the enzymatic reaction was performed in 20 mM glycine buffer (pH 8.6). Because of wide substrate specifity of the enzyme – it utilizes chlorinated as well as brominated substrates producing either non-absorbing chloride, or absorbing bromide ions – two different background electrolytes and the detection approaches were applied. A 10 mM chromate – 0.1 mM cetyltrimethylammonium bromide (pH 9.2) was used in combination with indirect detection and 20 mM ß-alanine – hydrochloric acid (pH 3.5) in combination with direct detection. The Michaelis constant (Km) of haloalkane dehalogenase for 1-bromobutane was determined by the developed EMMA methodology. The Km values 0.59 mM estimated by means of indirect detection method and 0.17 mM by means of direct detection method were comparable with the value 0.13 mM estimated previously by gas chromatography.