Determination of the Maximum Velocity of a Peroxidase-like Nanozyme

Nanozymes are functional nanomaterials with enzyme-like activities, which have good stability and specific nanoscale properties. Among them, peroxidase-like (POD-like) nanozymes with two substrates are the biggest chunk and have been widely applied in biomedical and environmental fields. Maximum velocity (V_max) is an essential kinetic parameter, accurate measurements of which can help in activity comparisons, mechanism studies, and nanozyme improvements. At present, the standardized assay determines the catalytic kinetics of POD-like nanozymes by a single fitting based on the Michaelis–Menten equation. However, the true V_max cannot be confirmed by this method due to the test condition that the concentration of a fixed substrate is finite. Here, a double fitting method to determine the intrinsic V_max of POD-like nanozymes is presented, which breaks through the limited concentration of the fixed substrate by an additional Michaelis–Menten fitting. Furthermore, a comparison of the V_max among five typical POD-like nanozymes validates the accuracy and feasibility of our method. This work provides a credible method to determine the true V_max of POD-like nanozymes, helping in activity comparisons and facilitating studies on the mechanism and development of POD-like nanozymes.