The multifaceted role of proteases and modern analytical methods for investigation of their catalytic activity.

We discuss the diverse functions of proteases in the context of their biotechnological and medical significance, as well as analytical approaches used to determine the functional activity of these enzymes. An insight into modern approaches to studying the kinetics and specificity of proteases, based on spectral (absorption, fluorescence), mass spectrometric, immunological, calorimetric, and electrochemical methods of analysis is given. We also examine in detail electrochemical systems for determining the activity and specificity of proteases.

Bielectrode Strategy for Determination of CYP2E1 Catalytic Activity: Electrodes with Bactosomes and Voltammetric Determination of 6-Hydroxychlorzoxazone.

We describe a bielectrode system for evaluation of the electrocatalytic activity of cytochrome P450 2E1 (CYP2E1) towards chlorzoxazone. One electrode of the system was employed to immobilize Bactosomes with human CYP2E1, cytochrome P450 reductase (CPR), and cytochrome (cyt ). The second electrode was used to quantify CYP2E1-produced 6-hydroxychlorzoxazone by its direct electrochemical oxidation, registered using square-wave voltammetry.

Electrochemical biosensor for trypsin activity assay based on cleavage of immobilized tyrosine-containing peptide.

In this work, we proposed a biosensor for trypsin proteolytic activity assay using immobilization of model peptides on screen-printed electrodes (SPE) modified with gold nanoparticles (AuNPs) prepared by electrosynthetic method. Sensing of proteolytic activity was based on electrochemical oxidation of tyrosine residues of peptides. We designed peptides containing N-terminal cysteine residue for immobilization on an SPE, modified with gold nanoparticles, trypsin-specific cleavage site and tyrosine residue as a redox label.

Biotransformation of phenytoin in the electrochemically-driven CYP2C19 system.

The possibility of the detection of atypical kinetic profiles of drug biotransformation using electrochemical systems based on immobilized cytochromes P450 with phenytoin hydroxylation by cytochrome P450 2C19 (CYP2C19) as an example was evaluated for the first time. For this purpose, we developed an electrochemical system, where one of the electrodes was modified by didodecyldimethylammonium bromide (DDAB) and was used as an electron donor for reduction of heme iron ion of the immobilized CYP2C19 and initiation of the catalytic reaction, while the second electrode was not modified and served for an electrochemical quantitation of 4-hydroxyphenytoin, which is a metabolite of antiepileptic drug phenytoin, by its oxidation peak. It was revealed that the dependence of the rate of 4-hydroxyphenytoin formation on phenytoin concentration is described by the equation for two enzymes or two binding sites indicating the existing of high- and low-affinity forms of the enzyme.

[The interactions of abiraterone and its pharmacologically active metabolite D4A with cytochrome P450 2C9 (CYP2C9)].

Interactions of cytochrome P450 2C9 (CYP2C9) were studied with the antitumor drug abiraterone and its pharmacologically active metabolite D4A, promising as an agent for prostate cancer treatment. It was shown by absorption spectroscopy, that both investigated compounds induced spectral changes of CYP2C9, indicating interactions of the pyridine nitrogen atom with the heme iron ion of the active site of the enzyme, but interactions of the ligands with the enzyme could be mediated by a water molecule bound to the heme iron ion. Based on the spectral changes, the values of dissociation constants (KS) for complexes of abiraterone and D4A with CYP2C9 were calculated as 1.