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Micellar electrokinetic chromatography: A convenient alternative to colorimetric and high performance liquid chromatographic detection to monitor protease activity
Author(s) -
Viglio Simona,
Zanaboni Giuseppe,
Cetta Giuseppe,
Iadarola Paolo,
Luisetti Maurizio,
Guglielminetti Maria
Publication year - 1998
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150191207
Subject(s) - micellar electrokinetic chromatography , chromatography , chemistry , capillary electrophoresis , high performance liquid chromatography , protease , capillary action , hydrolysis , electrokinetic phenomena , proteases , substrate (aquarium) , electrophoresis , enzyme , biochemistry , materials science , oceanography , composite material , geology
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 m M sodium tetraborate, pH 9.3, containing 65 m M 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 × 75 μm ID. This allowed us to determine the rate of hydrolysis of substrates and to calculate the kinetic parameters K m 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.