Determination of haloalkane dehalogenase enzymatic activity by capillary zone electrophoresis

GLATZ, Zdeněk, María Victoria MARINI PALOMEQUE, Michaela WIMMEROVÁ, Jiří DAMBORSKÝ and Yuji NAGATA. Determination of haloalkane dehalogenase enzymatic activity by capillary zone electrophoresis. Journal of Chromatography A. Netherlands: Elsevier, 2000, vol. 895, 1-2, p. 219-225. ISSN 0021-9606.

Basic information

• Original name: Determination of haloalkane dehalogenase enzymatic activity by capillary zone electrophoresis
• Authors: GLATZ, Zdeněk (203 Czech Republic, guarantor), María Victoria MARINI PALOMEQUE (858 Uruguay), Michaela WIMMEROVÁ (203 Czech Republic), Jiří DAMBORSKÝ (203 Czech Republic) and Yuji NAGATA.
• Edition: Journal of Chromatography A, Netherlands, Elsevier, 2000, 0021-9606.

Other information

• Original language: English
• Type of outcome: Article in a journal
• Field of Study: 10600 1.6 Biological sciences
• Country of publisher: Netherlands
• Confidentiality degree: is not subject to a state or trade secret
• WWW: URL
• Impact factor: Impact factor: 3.301
• RIV identification code: RIV/00216224:14310/00:00003315
• Organization unit: Faculty of Science
• Keywords in English: haloalkane dehalogenase; enzymatic activity; capillary zone electrophoresis
• Tags: Capillary Zone Electrophoresis, enzymatic activity, haloalkane dehalogenase

Abstract

A new sensitive method has been developed for the determination of haloalkane dehalogenase activity. The enzymatic reactions were carried out directly in thermostatted autosampler vials and the formation of product - bromide or chloride ions - was monito red by sequential capillary zone electrophoresis runs. The determinations were performed in a 75 mm fused-silica capillary using 5 mM chromate, 0.5 mM tetradecyltrimethylammonium bromide (pH 8.4) as a background electrolyte, separation voltage 15 kV (negative polarity ) and indirect detection at sample wavelength 315 nm, reference wavelength 375 nm for brominated and chlorinated substrates, respectively 0.1 M b-alanine-HCl (pH 3.50) as a background electrolyte, separation voltage 18 kV (negative polarity) and direct detection at 200 nm for brominated substrates. The temperature of capillary was in bot cases 25oC. The method is rapid, can be automated, and requires only small amount of enzyme preparation and substrate.

Links

• GA203/97/P149, research and development project: Name: Studium molekulárních mechanismů biodegradačních reakcí - konstrukce QSBR modelů a proteinové inženýrství dehalogenas

Investor: Czech Science Foundation, Study of the molecular mechanisms of biodegradation reactions - construction of QSBR models and protein engineering of haloalkane dehalogenases