*4.13. Reduction Potential*

Reduction potential of the CYP2C8 proteins was determined using safranin T as a redox probe as previously described [37]. Samples containing 5 μM of CYP2C8 variant, 20 nM paraquat (methyl viologen), 0.5 μM safarinin T, 10 mM EDTA, 50 μM of a 20% lipid reconstituted system [46], with or without 70 μM paclitaxel were prepared in 0.1 M potassium phosphate buffer, pH 7.4, in glass vials capped with septa. Samples were purged with N2 (g) for 20 min and then loaded into a Coy anaerobic glove box. 0.5 mL of each samples was loaded into UV–invisible plastic cuvettes stopped with a septa. Reduction potential was determined spectroscopically using a Cary 300 UV–vis spectrometer (Agilent Technologies). Cuvettes were equilibrated at 25 ◦C for each reading. Safranin T was used as the redox indicator to measure the reduction potential of the solution. Oxidation of the protein was monitored at 417 nm (reduction at 408 nm) and compared to the oxidation of safranin T at 535 nm. Reduction of the samples was initially achieved by irradiating samples on ice with time points up to 5 min with a 250 W tungsten lamp. Samples were further reduced by titrating anaerobic dithionite from 8 and 80 mM stocks. Re-oxidation was achieved by titrating anaerobic K3[Fe(CN)6] from 10 mM stocks. Spectral data were then processed using a MATLAB (R2014a) subroutine and analyzed using the Nernst equation as previously described [37].

**Supplementary Materials:** Supplementary materials can be found at http://www.mdpi.com/1422-0067/20/18/ 4626/s1.

**Author Contributions:** W.R.A., S.Z., D.D.M., and A.D. all contributed to the design of the experiments. W.R.A., S.Z., and K.S. conducted experiments. Mutations were made by D.D.M. W.R.A. and S.Z. analyzed the data and made figures. W.R.A., S.Z., and A.D. contributed to writing the manuscript. All authors accept the final version of the manuscript.

**Funding:** This project was supported by the American Heart Association [15SDG25760064], and in part by National Institutes of Health Grants R01 GM1155884 and R03 DA 04236502.

**Acknowledgments:** We thank Eric F. Johnson for the CYP2C8 gene. We thank Sligar for providing the MSPE3D1 gene. We thank Lucas Li at the Roy J. Carver Metabolomics Center at UIUC for mass spectrometry analysis.

**Conflicts of Interest:** The authors declare no conflict of interest.
