Oxygen penetration and diffusion into myoglobin revealed by quenching of zincprotoporphyrin IX fluorescence.

Oxygen quenching experiments were carried out on zincprotoporphyrin IX reconstituted myoglobin (MbFe-->Zn) at different temperatures and two solvent viscosities. The data were fit to a dynamic model for quenching of fluorophores in protein interiors previously presented (Biophysical J., 45 (1984) 789-794). The parameters associated with the oxygen entry rate (k+), exit rate (k-), and migration rate (chi) in the protein were obtained at six temperatures and two viscosities (1 and 8 cp), along with the activation enthalpies associated with the above rates (k+ and k-). The partition coefficient (alpha) was calculated at each temperature along with the free energy, delta G0, associated with this partition. The rate parameters (k+, k-, chi) and the partition coefficient (alpha) have also been determined for the sample in 40% sucrose (8 cp), to evaluate the effect of bulk solvent viscosities on these values. The steady-state Stern-Volmer quenching plot was calculated using the rate parameters obtained from the analysis (of the dynamic model). Comparison of the Stern-Volmer points obtained using the dynamic model and those obtained experimentally showed excellent agreement.