Capillary electrophoretic analysis of µ- and m-calpain using fluorescently labeled casein substrates

Abstract

Calpains are unique calcium-dependent thiol proteases that have been proposed to participate in a number of physiological processes including signal transduction and protein turnover in skeletal muscle. Calpains exist in two major forms. Interestingly, the two forms of protease show no significant difference in their action on various substrates. The only demonstrable difference in their activity involves the concentration of calcium required for activation. Both mu- and m-calpains typically achieve half maximal activation at 50 microM and 0.7 mM calcium, respectively. The focus of this study was to examine the action of both forms of calpain on casein substrates and assess whether any differences could be observed in the resulting peptide finger print using capillary electrophoresis. Purified mu- and m-calpain were incubated for various lengths of time with Oregon Green labeled alphas- and beta-casein. The reactions were stopped with sodium dodecyl sulfate (SDS) and products separated by capillary electrophoresis in micellar electrokinetic capillary chromatography (MEKC) mode using laser-induced fluorescence (LIF) detection. Comparison of the electropherograms showed no difference in the peptide profile for either enzyme. However, it was found that beta-casein was hydrolyzed more extensively than alphas-casein, by both enzymes. Capillary electrophoresis was found to be a very sensitive technique for detection of calpain activity. Using beta-casein as substrate, the CE approach was able to detect 2-3 ng of calpain activity. The results also suggest that capillary electrophoresis is a useful tool for proteolytic investigations of protein structure.

Capillary Electrophoresis for the Analysis of Biopolymers

“…Proteases. MECC was employed to examine the activity of both µ-calpain and m-calpain, which are calcium-dependent thiol proteases presumably participating in a number of physiological processes such as signal transduction (112). The two forms of calpains show no significant difference in their action on various substrates.…”

Microscale separation methods for enzyme kinetics assays

“…Figure 1 illustrates the typical outcome of an off-line MEKCbased enzyme assay using direct UV detection. The other popular detection technique is laser-induced fluorescence (LIF) with fluorescently-labeled substrates [28,31,32,34,44,45,64]. This technique is particularly advantageous in studies in which very small amounts of substrates or products are to be detected, because the detection limits of the LIF technique are often comparable with those of mass spectrometric methods.…”