Methods of Measuring Enzyme Activity Ex Vivo and In Vivo

Abstract

Enzymes catalyze a variety of biochemical reactions in the body and, in conjunction with transporters and receptors, control virtually all physiological processes. There is great value in measuring enzyme activity ex vivo and in vivo. Spatial and temporal differences or changes in enzyme activity can be related to a variety of natural and pathological processes. Several analytical approaches have been developed to meet this need. They can be classified broadly as methods either based on artificial substrates, with the goal of creating images of diseased tissue, or based on natural substrates, with the goal of understanding natural processes. This review covers a selection of these methods, including optical, magnetic resonance, mass spectrometry, and physical sampling approaches, with a focus on creative chemistry and method development that make ex vivo and in vivo measurements of enzyme activity possible.

Processing, Characteristics and Composition of Umqombothi (a South African Traditional Beer)

“…Automated online analysis, together with different computer software programs that provide predefined bioprocess optimisation strategies, versatile sample processing, and feed control, is improving the management of complex dynamic biological systems [89]. Other online platforms allow for the ex vivo measurement of extracellular and intracellular enzyme activity, while making the quantitative determination of metabolic flux dynamics possible [89,90]. Furthermore, bioprocess development, especially in biotechnology, has experienced the replacement of empirical strategies by new systematic protocols of experimentation, with better reproductivity and analytical precision accuracy [89].…”

“…The importance of RSM, despite its limitations, must not be underestimated, since it is still one of the most effective methods improving fermentation media, synthetic or otherwise [88][89][90]. With a smaller number of variables, RSM can produce a highly accurate response (or output) in non-linear optimisation [90,91].…”

“…The importance of RSM, despite its limitations, must not be underestimated, since it is still one of the most effective methods improving fermentation media, synthetic or otherwise [88][89][90]. With a smaller number of variables, RSM can produce a highly accurate response (or output) in non-linear optimisation [90,91]. In addition, RSM can generate a mathematical model in the form of a 3D plot or a second-order polynomial equation, both useful in industrial applications [88,89,97,98].…”

Molecular Structure, Binding Affinity, and Biological Activity in the Epigenome

“…In vivo activity tests cover investigations on living animals with various approaches. For example, in vivo enzyme activity tests can be performed by magnetic resonance (MRI)-based methods, microdialysis and fluorescence imaging [164] among others. In vivo MRI-based methods can apply microinjection of contrast agents cleaved by a specific enzyme to map the gene expression of transgenic animals, and improve our knowledge of mRNA expression, inheritance patterns and plasmid gene expression [165].…”

Coumarin-Based Profluorescent and Fluorescent Substrates for Determining Xenobiotic-Metabolizing Enzyme Activities In Vitro

“…Fluorescence assays are also amenable to miniaturization of the reaction setup and high throughput. Thus, further increases in sensitivity can be achieved with a decrease in demand for enzyme source and fluorescent substrate [ 13 , 14 , 15 ]. An advantage of fluorophore-based enzyme assays is also that both fixed time and online continuous assays (milliseconds to hours) can be applied for multiple purposes.…”