For example, Fig. 2 shows time-resolved courses of the Trp fluorescence changing during refolding of mutant form m2 of apomyoglobin. It is seen that at zero time t0 the intensity values of Trp fluorescence at 335 nm are different. This is an indication that within the dead time of the instrument the intermediate state is accumulated. So, at final urea concentrations below 3 M, there are two consecutive refolding phases: The first phase occurs within the dead time of a stopped-flow instrument and is revealed by a jump-wise increase of fluorescence intensity, and the second phase is observed as a slow decrease of fluorescence intensity. At final urea concentrations above 3 M, there is only one fast phase, which manifests itself as a burst-like insignificant increase of fluorescence intensity. So, owing to the instrument dead time, it is only the result of the fast phase that can be observed. After the protein, refolding is completed the fluorescence intensity values correspond to the equilibrium values. It should be noted that the kinetic intermediate I has a higher fluorescence intensity than that of the native N or unfolded state U. This property of the intermediate state is used to separate the kinetic transition U«I from the transition I«N. Since the slow phase of apomyoglobin refolding always leads to a decrease of fluorescence intensity, folding into the native state is believed to start from the intermediate state. At a given urea concentration M, the transient intermediate state population fI can be calculated from the burst phase amplitude A. Baryshnikova et al. described in detail the approach allowing calculating the dependence of the population of the apomyoglobin intermediate state fI on the urea concentration. The gist of the method is that the population of a rapidly formed intermediate state affects the amplitude of the subsequent slow kinetics of folding. For example, Fig. 2 demonstrates the population of the molten globule state fI of mutant form m2 of apomyoglobin calculated from the burst phase amplitude kinetic curves in Fig. 2 according to Equation 5. The kinetics of refolding for all mutant forms of apomyoglobin were measured and the populations of the molten globule state were calculated. Fig. 3 shows dependencies of populations fI of the molten globule state versus urea concentration for all mutant forms of apomyoglobin with substitutions of hydrophobic amino acid residues on the protein surface and in its hydrophobic core. Table 1 lists values of urea concentration corresponding to the midpoint of transition for wild type apomyoglobin and its mutant forms. As can be seen, none of these substitutions have effect on the stability of the molten globule state. But all mutations change the stability of the native state of apomyoglobin. This can be concluded from the curves of equilibrium unfolding of the mutant forms of apomyoglobin.