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. Author manuscript; available in PMC: 2019 Sep 1.
Published in final edited form as: Arch Biochem Biophys. 2018 Jun 26;653:90–96. doi: 10.1016/j.abb.2018.06.014

Fig. 2. Absorbance spectra of the ligand-free unlabeled, STM-labeled, and PGS-treated F215C mutant of CYP3A4.

Fig. 2.

To demonstrate that unconjugated STM was likely removed during the washing step and is not responsible for the high-spin shift (390 nm peak), a mock-bioconjugation control experiment was performed by treating the F215C mutant with non-modified progesterone (PGS, a good mimic of STM) under similar conditions. As evident from the absorbance spectra, the PGS-treated CYP3A4 spectrally resembles the untreated P450. Thus, the high-spin shift in STM-labeled CYP3A4 is induced by the attached steroid rather than the trace amounts of non-conjugated STM that might be present in the sample.