Colorimetric Assay for Protein Redox Activity – a Complement to Electrochemistry

Abstract

Oxidoreductases are vital biocatalytic tools for driving redox conversions that are challenging using conventional catalysts. Monooxygenases, an important class of these enzymes, have been the subject of enzyme engineering due to their high activity and versatile substrate scope. Reactions performed by these biocatalysts have long been monitored by a colorimetric method involving the coupling of a dye precursor to naphthalene hydroxylation products generated by the enzyme. Despite the popularity of this method, we found the dye product to be unstable, preventing quantitative readout. By incorporating an extraction step to solubilize the dye produced, we have improved this assay to the point where quantitation of enzyme activity is possible. Further, by incorporating spectral deconvolution, we have, for the first time, enabled independent quantification of the two possible regioisomeric products: 1-naphthol and 2-naphthol. Previously, such analysis was only possible with chromatographic separation, increasing the cost and complexity of analysis. The efficacy of our improved workflow was evaluated by monitoring the activity of a toluene-4-monooxygenase enzyme from Pseudomonas mendocina KR-1. Our colorimetric regioisomer quantification was found to be consistent with chromatographic analysis by HPLC. The development and validation of a quantitative colorimetric assay for monooxygenase activity that enables regioisomeric distinction and quantification represents a significant advance in analytical methods to monitor enzyme activity. By maintaining facile, low-cost, high-throughput readout while incorporating quantification, this assay represents an important alternative to more expensive chromatographic quantification techniques. For the electrochemistry community, the use of monooxygenases or other redox capable enzymes for biocatalysis must be complemented by a toolkit of methods for accurate enzyme activity measurement. Our approach demonstrates that accurate, mixture analysis is possible in a higher throughput using the improved colorimetric method. The relevant assay reaction, the hydroxylation of naphthalene, may be readily incorporated in the future for measurement schemes with other enzymes than the tested toluene-4-monooxygenase, thus enabling opportunities to tune these biocatalysts towards desired activities.