Skip to main content
. Author manuscript; available in PMC: 2013 Aug 14.
Published in final edited form as: Biochemistry. 2012 Jul 31;51(32):6441–6457. doi: 10.1021/bi300744z

Figure 2.

Figure 2

Optical titrations and binding isotherms for nitrogen heterocycles binding to CYP2C9. 1,2,3- TRZs binding to CYP2C9 yield type II behavior. UV/Vis equilibrium absorbance titration (20°C) of purified CYP2C9h (1–2μM; 0.1 M KPi, pH = 7.4, + 20% glycerol) with 1,2,3-TRZ (top), 1,2,4-TRZ (middle), and IMZ (bottom). Shown are calculated difference spectra (left), with the α and β region of the recorded absolute spectra as inset, and the resultant binding isotherms (right), generated by plotting Δ(peaktrough) intensities as a function of ligand concentration, with best fits used to estimate KD (Table 2). As with CYP3A4, 1,2,4-TRZ displays complex equilibrium binding to CYP2C9, which is most evident as curvature in the Eadie-Hofstee transforms (inset). The data fit best to a two site model (Material and Methods, equation 4) where binding at each site induces similar spin-shifts.