Advances in enzyme substrate analysis with capillary electrophoresis

Abstract

Capillary electrophoresis provides a rapid, cost-effective platform for enzyme and substrate characterization. The high resolution achievable by capillary electrophoresis enables the analysis of substrates and products that are indistinguishable by spectroscopic techniques alone, while the small volume requirement enables analysis of enzymes or substrates in limited supply. Furthermore, the compatibility of capillary electrophoresis with various detectors makes it suitable for KM determinations ranging from nanomolar to millimolar concentrations. Capillary electrophoresis fundamentals are discussed with an emphasis on the separation mechanisms relevant to evaluate sets of substrate and product that are charged, neutral, and even chiral. The basic principles of Michaelis-Menten determinations are reviewed and the process of translating capillary electrophoresis electropherograms into a Michaelis-Menten curve is outlined. The conditions that must be optimized in order to couple off-line and on-line enzyme reactions with capillary electrophoresis separations, such as incubation time, buffer pH and ionic strength, and temperature, are examined to provide insight into how the techniques can be best utilized. The application of capillary electrophoresis to quantify enzyme inhibition, in the form of KI or IC50 is detailed. The concept and implementation of the immobilized enzyme reactor is described as a means to increase enzyme stability and reusability, as well as a powerful tool for screening enzyme substrates and inhibitors. Emerging techniques focused on applying capillary electrophoresis as a rapid assay to obtain structural identification or sequence information about a substrate and in-line digestions of peptides and proteins coupled to mass spectrometry analyses are highlighted.

Clinical and pharmaceutical applications of affinity ligands in capillary electrophoresis: A review

“…Enzymes are another set of biological agents that have been used in CE [105][106][107][108][109][110][111][112][113][114]. Enzymes are biological molecules that act as catalysts for chemical reactions in living systems.…”

“…One application in CE that has already been mentioned in Section 5 is use of the enzyme cellulase as a stereoselective binding agent for chiral separations [59,74]. However, CE has also been used in a number of studies to characterize enzyme-substrate interactions and kinetics [105][106][107][108][109][110][111][112]. This has been done by using pre-capillary assays, in-capillary enzyme assays or immobilized enzyme reactors (IMERs) [105].…”

“…In a pre-capillary assay, the enzyme and substrate are mixed and incubated with the co-factors and other reaction components for a given period of time [105]. The reaction is then quenched by freezing or by adding perchloric acid or hydrochloric acid [106][107][108][109].…”

“…6. General scheme for an in-capillary enzyme assay conducted by CE [105,112]. mixture is injected, this format for an enzyme assay requires much more reagent and sample than an in-capillary method because the reaction components are combined and incubated before they are used in CE [110,113,114].…”

Solid acids assisted matrix solid‐phase dispersion microextraction of alkaloids by capillary electrophoresis coupled with quadrupole time‐of‐flight mass spectrometry

“…The successful analytical procedure relies on appropriate sample preparation methods and sensitive analytical instruments . CE is an efficient separation technique widely applied to the various fields such as enzyme inhibitor screening , enzyme substrate , vitamins , pharmaceutical analysis, and drug screening and so on. To date, CE has unique dominant positions thanks to automation, rapid analysis (2–30 min), minimal sample requirement (2–10 µL), and high separation efficiency (resolution 1.35–1.45) .…”