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 (Vmax) 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 Vmax 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 Vmax 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 Vmax among
five typical POD-like nanozymes validates the accuracy and feasibility
of our method. This work provides a credible method to determine the
true Vmax of POD-like nanozymes, helping
in activity comparisons and facilitating studies on the mechanism
and development of POD-like nanozymes.