Kinetic Analysis of VAP-1 Enzyme Activity

Putative, naturally occurring substrates of human VAP-1 were selected based on inclusion in the human metabolome database V.2.5 (http://www.hmdb.ca), and with reference to Shen et al,20 (link) specifically cysteamine, methylamine, dopamine, ethylamine and phenethylamine (Sigma). A 96-well plate was loaded with 50 ng recombinant VAP-1 (Biolegend) in 100 µL Dulbecco's phosphate-buffered saline with increasing concentrations of test substrate supplemented to test wells. A H₂O₂ standard curve was created, and enzyme activity quantified using the Amplex-UltraRed detection system across increasing concentrations of test substrate for a fixed concentration of VAP-1 under atmospheric conditions. Kinetic parameters were estimated by Michaelis-Menten methodology (see online supplementary figure 1). Benzylamine served as an internal positive control.