Analysis of activity and inhibition of oxygen-dependent enzymes by optical respirometry on the LightCycler system

There is currently a need for a method capable of measuring the activity and inhibition of biologically relevant oxygenases in a format that enables the convenient, fast, and cost-efficient generation of dose-response information. Here we describe a low-volume luminescence-based assay for the measurement of such oxygen-dependent enzymes. The assay employs a photoluminescent oxygen-sensitive probe and glass capillary microcuvettes measured on the Roche LightCycler detection platform. Three discrete types of oxygen probe were evaluated for this application: (i) solid-state coatings, (ii) soluble macromolecular MitoXpress probe, both phosphorescent porphyrin-based, and (iii) a luminescent Ir(III)-based nanoparticle probe. Measurement parameters were optimised and subsequently applied to the analysis of three biologically relevant oxygenases, namely cytochrome P450 (CYP), monoamine oxygenase (MAO), and cyclooxygenase (COX). CYP enzymes are central players in drug detoxification while specific inhibitors of MAO and COX are important for therapeutic intervention and treatment of neurological and inflammatory diseases, respectively. To determine assay utility, oxygen consumption catalysed by all three enzyme types was measured and the effect of specific inhibitors determined. The panel included the MAO-A/B inhibitors clorgyline, toloxatone, deprenyl, and the COX-1/2 inhibitors niflumic acid, nimesulide, SC-560, ketoprofen, and phenylbutazone. IC₅₀ values were then compared with literature values. The measurement methodology described allows the low-volume analysis of biologically relevant oxygenases and displays the requisite sensitivity and throughput to facilitate routine analysis. It is also applicable to other O₂-dependent enzymes and enzymatic systems.