Study of enzymatic reaction by electrophoretically mediated microanalysis in a partially filled capillary with indirect or direct detection.
Electrophoretically mediated microanalysis (EMMA), in combination with a partial filling technique and indirect or direct detection, is described for the study of enzymes reacting with the high mobility inorganic or organic anions as substrates or products. Part of the capillary is filled with a buffer optimized for the enzymatic reaction, the rest of the capillary with the background electrolyte being optimal for the separation of substrates and products. With haloalkane dehalogenase, chosen as a model enzyme, the enzymatic reaction was performed in a 20 mM glycine buffer (pH 8.6). Because of the wide substrate specificity of this enzyme, utilizing chlorinated as well as brominated substrates and producing either nonabsorbing chloride or absorbing bromide ions, two different background electrolytes and detection approaches were adopted. A 10 mM chromate-0.1 mM cetyltrimethylammonium bromide background electrolyte (pH 9.2) was used in combination with indirect detection and 20 mM beta-alanine-hydrochloric acid (pH 3.5) in combination with direct detection. The Michaelis constant (K(m)) of haloalkane dehalogenase for 1-bromobutane was determined. The K(m) 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.
Telnarová
Magdaléna
M
Department of Biochemistry, Masaryk University, Brno, Czech Republic.
Vytisková
Sona
S
Chaloupková
Radka
R
Glatz
Zdenek
Z
D019075:Electrophoresis, Capillary / Q000379:methods*; D004798:Enzymes / Q000032:analysis* / Q000378:metabolism*; D006867:Hydrolases / Q000032:analysis / Q000378:metabolism; D007700:Kinetics; D008832:Microchemistry / Q000379:methods*; D020578:Sphingomonas / Q000201:enzymology
D004798:Enzymes; D006867:Hydrolases; C046137:haloalkane dehalogenase