Measuring peroxidasin activity in live cells using bromide addition for signal amplification

Abstract

Peroxidasin (PXDN) is involved in the crosslinking of collagen IV, a major constituent of basement membranes. Disruption of basement membrane integrity as observed in genetic alterations of collagen IV or PXDN can result in developmental defects and diverse pathologies. Hence, the study of PXDN activity in (patho)physiological contexts is highly relevant. So far, measurements of PXDN activity have been reported from purified proteins, cell lysates and de-cellularized extracellular matrix. Here, for the first time we report the measurement of PXDN activity in live cells using the Amplex Red assay with a signal amplifying modification. We observe that bromide addition enhances the obtained signal, most likely due to formation of HOBr. Abrogation of signal amplification by the HOBr scavenger carnosine supports this hypothesis. Both, pharmacological inhibition as well as complementary genetic approaches confirm that the obtained signal is indeed related to PXDN activity. We validate the modified assay by investigating the effect of Brefeldin A, to inhibit the secretory pathway and thus the access of PXDN to the extracellular Amplex Red dye. Our method opens up new possibilities to investigate the activity of PXDN in (patho)physiological contexts.

Keywords

• Peroxidasin
• Amplex red
• Hypobromous acid
• Carnosine
• Brefeldin A