Micellar electrokinetic chromatography: a convenient alternative to colorimetric and high performance liquid chromatographic detection to monitor protease activity.
High performance capillary electrophoresis (HPCE) has been exploited as an analytical method alternative to current procedures for the determination of proteolytic activity of elastases from different sources. Due to some drawbacks with capillary zone electrophoresis (CZE), the mode of operation employed for the assay of elastolytic activity was micellar electrokinetic chromatography (MEKC). Using a background electrolyte consisting of 35 mM sodium tetraborate, pH 9.3, containing 65 mM SDS and 15% v/v methanol, separation of intact peptide substrate from products of proteolytic reaction was easily achieved in a fused-silica capillary of 50 cm effective length x 75 microm ID. This allowed us to determine the rate of hydrolysis of substrates and to calculate the kinetic parameters Km and k(cat) of the proteases investigated. A comparison of these data with those obtained from high performance liquid chromatography (HPLC)-based experiments showed that MEKC is a convenient technique for studying protease kinetics.
Dipartimento di Biochimica A. Castellani, Università di Pavia, Italy.
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S
Viglio
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G
Zanaboni
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M
Luisetti
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G
Cetta
M
M
Guglielminetti
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P
Iadarola
D001232:Aspergillus fumigatus / Q000201:enzymology; D001881:Borates; D002851:Chromatography, High Pressure Liquid; D020374:Chromatography, Micellar Electrokinetic Capillary / Q000379:methods*; D003124:Colorimetry; D010450:Endopeptidases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010196:Pancreatic Elastase / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D012680:Sensitivity and Specificity
D001881:Borates; D010446:Peptide Fragments; C010634:sodium borate; D010450:Endopeptidases; D010196:Pancreatic Elastase