P450 enzymes are ubiquitous in nature and carry out a wide range of important reactions including the activation of carbon centers for catabolism, steroid metabolism, and detoxification of xenobiotics (1). Due to the important physiological functions of P450 enzymes, the reaction intermediates of P450 chemistry have been a subject of investigation for many years. Several discreet steps occur in the hydroxylation chemistry of the cytochrome P450s including: substrate binding, first electron transfer to the P450 from a physiological redox partner, oxygen binding (to form oxy-P450), a second electron transfer event (to form a reduced oxy or peroxo state), proton transfer to the distal oxygen (forming hydroperoxo), and an oxygen scission event producing a putative high valent iron-oxo species that subsequently generates hydroxylated product (2). The high valent iron-oxo has long been thought to be analogous to the activated iron species characterized in other oxidative enzymes (horseradish peroxidase (HRP), 1 catalase, and chloroperoxidase (CPO)) and referred to as Compound I, a ferryl-oxo-() porphyrin cation radical (3-5). The intermediates in the complex P450 reaction cycle have been gradually divulged through the use of a wide range of spectroscopic techniques (6 -8) and the use of methods that allow access to the fast steps occurring after O 2 binding (2, 9). Recently the peroxo and hydroperoxo intermediates have been observed through the use of cryoenzymology techniques combined with radiolysis, generating these intermediates through gradual temperature annealing (2, 10). Although there has been no identification of a Compound I intermediate by these techniques, it has been inferred by indirect measurements (2,11,12). The Compound I state as well as the peroxo and hydroperoxo states of the enzyme have been proposed to be active in oxygenation events (13,14).To probe the nature of Compound I, meta-chloroperoxybenzoic acid (m-CPBA) has been used as an oxidizing agent to produce this high valent iron-oxo through heterolytic cleavage of the organic peroxide in CPO and HRP. The Compound I species of CPO and HRP have sufficient half-lives for resonance Raman and EPR studies and have been well characterized (3,5,(15)(16)(17). The same peroxy acid and its derivatives have been used with P450s in attempts to generate intermediates. Evidence for the existence of Compound I in P450s was demonstrated through substrate oxygenation or hydroxylation in the reaction of various organic peroxides with either liver microsomal P450 or CYP101, respectively (18,19,21).Contrary to the evidence for Compound I formation in cytochrome P450 using m-CPBA, Blake and Coon (20) reported that the reactions of several peroxy acids with CYP2B4 generated other active species, which are neither Compound I nor its one-electron-reduced adduct (Compound II). However, in previous studies the generation of the Compound I intermediate in cytochrome P450 (CYP101) required a large excess of peroxyacid, leading to the rapid formation, then conversion of ...
Limited training, high cost, and low equipment mobility leads to inaccuracies in decision making and is concerning with serious ocular injuries such as suspected ruptured globe or post-operative infections. Here, we present a novel point-of-service (POS) quantitative ascorbic acid (AA) assay with use of the OcuCheck Biosensor. The present work describes the development and clinical testing of the paper-based biosensor that measures the changes in electrical resistance of the enzyme-plated interdigitated electrodes to quantify the level of AA present in ocular fluid. We have demonstrated the proof-of-concept of the biosensor testing 16 clinical samples collected from aqueous humor of patients undergoing therapeutic anterior chamber paracentesis. Comparing with gold standard colorimetric assay for AA concentration, OcuCheck showed accuracy of >80%, sensitivity of >88% and specificity of >71%. At present, there are no FDA-approved POS tests that can directly measures AA concentration levels in ocular fluid. We envisage that the device can be realized as a handheld, battery powered instrument that will have high impact on glaucoma care and point-of-care diagnostics of penetrating ocular globe injuries.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.