The thioredoxin redox potential and redox charge are surrogate measures for flux in the thioredoxin system.

The thioredoxin system plays a central role in intracellular redox regulation and its dysregulation is associated with a number of pathologies. However, the connectivity within this system poses a significant challenge for quantification and consequently several disparate measures have been used to characterize the system. For in vitro studies, the thioredoxin system flux has been measured by NADPH oxidation while the thioredoxin redox state has been used to estimate the activity of the system in vivo. The connection between these measures has been obscure although substrate saturation in the thioredoxin system results from the saturation of the thioredoxin redox cycle. We used computational modeling and in vitro kinetic assays to clarify the relationship between flux and the current in vivo measures of the thioredoxin system together with a novel measure, the thioredoxin redox charge (reduced thioredoxin/total thioredoxin). Our results revealed that the thioredoxin redox potential and redox charge closely tracked flux perturbations showing that these indices could be used as surrogate measures of the flux in vivo and, provide a mechanistic explanation for the previously observed correlations between thioredoxin oxidation and certain pathologies. While we found no significant difference in the linear correlations obtained for the thioredoxin redox potential and redox charge with the flux, the redox charge may be preferred because it is bounded between zero and one and can be determined over a wider range of conditions allowing for quantitative flux comparisons between cell types and conditions.

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.