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.

过氧化物酶 (PXDN) 参与 IV 型胶原蛋白的交联，IV 型胶原蛋白是基底膜的主要成分。 IV 型胶原或 PXDN 基因改变中观察到的基底膜完整性破坏可能导致发育缺陷和多种病理。因此，（病理）生理背景下 PXDN 活性的研究具有高度相关性。到目前为止，已经报道了从纯化的蛋白质、细胞裂解物和去细胞的细胞外基质中测量 PXDN 活性。在这里，我们首次报告使用经过信号放大修改的 Amplex Red 测定法测量活细胞中的 PXDN 活性。我们观察到溴化物的添加增强了获得的信号，这很可能是由于 HOBr 的形成。 HOBr 清除剂肌肽消除信号放大支持了这一假设。药理学抑制以及互补的遗传方法均证实所获得的信号确实与 PXDN 活性相关。我们通过研究布雷菲德菌素 A 的作用来验证修改后的测定法，以抑制分泌途径，从而抑制 PXDN 与细胞外 Amplex Red 染料的接触。我们的方法为研究 PXDN 在（病理）生理环境中的活性开辟了新的可能性。