The thioredoxin system and not the Michaelis-Menten equation should be fitted to substrate saturation datasets from the thioredoxin insulin assay.

INTRODUCTION: The thioredoxin system, consisting of thioredoxin reductase, thioredoxin and NADPH, is present in most living organisms and reduces a large array of target protein disulfides. OBJECTIVE: The insulin reduction assay is commonly used to characterise thioredoxin activity in vitro, but it is not clear whether substrate saturation datasets from this assay should be fitted and modeled with the Michaelis-Menten equation (thioredoxin enzyme model), or fitted to the thioredoxin system with insulin reduction described by mass-action kinetics (redox couple model). METHODS: We utilized computational modeling and in vitro assays to determine which of these approaches yield consistent and accurate kinetic parameter sets for insulin reduction. RESULTS: Using computational modeling, we found that fitting to the redox couple model, rather than to the thioredoxin enzyme model, resulted in consistent parameter sets over a range of thioredoxin reductase concentrations. Furthermore, we established that substrate saturation in this assay was due to the progressive redistribution of the thioredoxin moiety into its oxidised form. We then confirmed these results in vitro using the yeast thioredoxin system. DISCUSSION: This study shows how consistent parameter sets for thioredoxin activity can be obtained regardless of the thioredoxin reductase concentration used in the insulin reduction assay, and validates computational systems biology modeling studies that have described the thioredoxin system with the redox couple modeling approach.