Quantitative analysis of H₂O₂ transport through purified membrane proteins

Hydrogen peroxide (H₂O₂) is an important signal molecule produced in animal and plant cells. The balance of H₂O₂ between the intra- and extracellular space is regulated by integral membrane proteins, which thereby modulate signaling. Several methods have been established to analyze aquaporin mediated transport of H₂O₂ in whole cells with the intrinsic limitation that the amount of protein responsible for a certain activity cannot be standardized. As a consequence, the quantification of the transport and specific activity is difficult to extract making it problematic to compare isoforms and mutated variants of one specific target. Moreover, in cell-based assays, the expression of the target protein may alter the physiological processes of the host cell providing a complication and the risk of misleading results. To improve the measurements of protein based H₂O₂ transport, we have developed an assay allowing quantitative measurements.•Using purified aquaporin reconstituted in proteoliposomes, transport of H₂O₂ can be accurately measured.•Inside the liposomes, H₂O₂ catalyzes the reaction between Amplex Red and horseradish peroxidase (HRP) giving rise to the fluorescent product resorufin.•Analysing pure protein provides direct biochemical evidence of a specific transport excluding putative cellular background.