![]() Biochemical character- ization indicated that preparations of enzyme contained pri- marily palmitic acid with some stearic acid. Crystallization of P450 2C8-Ligand Complexes -P450 2C8 was crystallized originally as a dimer with two fatty acid molecules bound at the dimer interface (15). The concentration-dependent changes in absorbance were fit with a quadratic form of the binding equation by non- linear regression using SlideWrite Plus (Advanced Graphics Software, Carlsbad, CA) as described under “Results.” Addi- tional models of multiple equilibrium binding were fit to the data by numerical simulations and nonlinear regression using DynaFit 4.02.089 (BioKin Ltd.) (21). The conversion of the enzyme from low spin to high spin was monitored by the increase in absorbance of the enzyme at 646 nm and decrease at 570 nm. Following correction of each spectrum for dilution, difference spectra were computed by subtraction of the spectrum of the enzyme obtained in the absence of the ligand from each of the spectra recorded following the addition of the ligand. The final concentration of methanol was Յ 2%. ![]() was added in 0.5- l aliquots to the sample cuvette, and spectra were recorded digitally at 0.25-nm intervals from 260 to 700 nm.
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