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.

Abstract licence: CC BY-NC

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