of consistency, amounts of enzyme should be normalized to equivalent
amounts of nanomole P450 per incubation (Williams et al., 2002b). Suppliers
often provide ready made buffers, or these can be made quite simply in the
laboratory. Preincubation of enzyme, substrate, and buffer at 37C with
shaking should ensure the initial reaction velocity is near its theoretical
maximum when the reaction is initiated by the addition of cofactor
FIGURE 15.3 Assessment of time linearity using (a) substrate depletion and (b)
metabolite formation approaches and (c) confirmation of linearity with respect to
protein for the metabolite formation approach.
494 REACTION PHENOTYPING