Quantification of spermine oxidase (SMOX) activity in tissues by HPLC

The polyamine oxidases, SMOX and PAOX, are enzymes involved in the normal metabolism of polyamines. Both enzymes are implicated in numerous human diseases including cancer, reperfusion injury, and neurodegenerative diseases. The ability to directly measure the activity of these enzymes is imperative in understanding their role in human health and disease. Most assays currently used to measure both SMOX and PAOX activity use a coupled reaction with horse radish peroxidase (HRP). These assays cannot be used when evaluating certain compounds for potential polyamine oxidase inhibition if these compouds also affect HRP activity. Additionally, since most assays use H₂O₂ production as an indicator of oxidase activity they cannot be used to evaluate polyamine oxidase activity in the presence of iron or other divalent metals. This prevents the use of these assays to evaluate polyamine oxidase activity in tissue samples. Here we describe the protocols for determining polyamine oxidase activity in an HRP-independent manner via an HPLC-based assay allowing for evaluation of both compounds that may interfere with HRP activity and polyamine oxidase activity in tissue samples.