Micellar electrokinetic chromatography: A convenient alternative to colorimetric and high performance liquid chromatographic detection to monitor protease activity

Abstract

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.

Development of off‐line and on‐line capillary electrophoresis methods for the screening and characterization of adenosine kinase inhibitors and substrates

“…CE has recently emerged as a powerful tool for enzyme analysis [14][15][16], including the determination of Michaelis-Menten constants [17][18][19][20], and for enzyme inhibition assays [18][19][20][21][22] exhibiting a number of advantages over conventional methods. These include rapid separation, the ability to simultaneously discriminate between, and identify multiple components of similar structure with high resolution [23], ultralow sample volume requirements, and high-throughput by automation.…”

A highly sensitive CE‐UV method with dynamic coating of silica‐fused capillaries for monitoring of nucleotide pyrophosphatase/phosphodiesterase reactions

“…An alternative method for assaying enzyme activity with great potential for drug screening is capillary electrophoresis (CE) [20]. Recently, we have developed CE-based enzyme assays for the determination of Michaelis-Menten constants (K m values), maximal velocities (V max ) for substrates, and inhibition constants (IC 50 or K i values) for enzyme inhibitors of various nucleotide and nucleoside-metabolizing enzymes, namely ecto-5 0 -nucleotidase [21], nucleoside triphosphate diphosphohydrolase (ecto-NTPDase) [22], adenosine kinase [23], and herpes simplex type 1 (HSV-1) thymidine kinase [24].…”

Diagnosis of late-infantile neuronal ceroid lipofuscinosis: A new sensitive method to assay lysosomal pepstatin-insensitive proteinase activity in human and animal specimens by capillary electrophoresis

“…Recent advances in CE have indicated that this technique has become the method of choice for determining numerous proteolytic activities in vitro and in vivo [18,19,21,[26][27][28]. To investigate whether the applicability of CE in free solution or MEKC can be further extended to measure TPP-I activity, human skin fibroblasts cultures were considered as a source of this proteinase.…”

“…Although presently used, both the above-cited methods are still rather laborious and time-consuming. In this respect, because capillary electrophoresis (CE) has proven to be an excellent tool for monitoring activities of serine-and metallo-proteinases [18][19][20][21], we explored the possibility of using this technique to assay for TPP-I. This paper describes (i) a systematic investigation carried out with capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) to optimize the experimental conditions for monitoring TPP-I activity using a synthetic peptide, (ii) the determination of TPP-I activity using various human and animal samples as sources of this ezyme, and (iii) the determination of TPP-I activity in human specimens obtained from patients affected by LINCL.…”