The assumption is then made that the first reaction (the activation step) is pseudo-first order (PFO) . However, it has been demonstrated that an excessive concentration of E 1 is not sufficient to guarantee the validity of PFO model. Instead, it is necessary that initial concentration of zymogen for (1) be much less than the Michaelis constant of the primary reaction . Thus, from an experimental point of view, it is difficult to ensure the validity of the PFO model when the Michaelis constant is unknown.