Figure 3.

Estradiol-induced spectral changes of CcO. (A) Difference spectra induced by addition of 0.5 mM (1–4) or 1 mM (5–7) estradiol benzoate to the air oxidized CcO (1.22 µM). Spectra were recorded at the indicated time intervals after the hormone addition. The basic medium (pH 8.0) was supplemented with 13 mM DM. (B) Effect of cyanide on the estradiol-induced spectral shift of heme a₃³⁺. Spectrum 1 (black)—control (difference spectrum recorded 3 min after addition of 1 mM estradiol to the air oxidized CcO). Spectrum 2 (red) was obtained as 1 but with the cyanide-complexed CcO (produced by 1 h incubation of CcO in the assay medium supplemented with 5 mM KCN and 25 µM potassium ferricyanide which prevented oxidized cyanide adduct from slow reduction). Spectrum 3 (blue)—as 2 but recorded 3 h after estradiol addition. Other conditions are as in panel A. (C) Time dependence of the estradiol-induced spectral changes. Traces 1 (black circles) and 2 (red triangles) represent time evolution of absorption difference at (437–415 nm) caused in the ligand-free oxidized CcO by incubation with 0.5 mM and 1 mM estradiol, respectively (see panel A). Trace 3 (blue triangles)—development of absorption difference at (428–450 nm) induced in the cyanide-complexed CcO by incubation with 1 mM estradiol (see panel B).