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. 2020 Dec 3;48(22):12845–12857. doi: 10.1093/nar/gkaa1147

Figure 2.

Figure 2.

Initial spectroscopic characterization and chromophore analysis of NewPHLs. (A) Initial UV–vis spectra after purification for DsNewPHL (solid black line) and after in vitro oxidation of the protein bound FAD with potassium ferricyanide (solid blue line). The spectra for MmNewPHL are shown in dotted lines, after purification (red dotted) and with FADox after oxidation (green dotted). Spectra are normalized at 630 nm for FADH° containing samples and at 375 nm for the NewPHLs with only FADox. (A, inlay) Cofactor analysis via thin layer chromatography (TLC) of heat denatured DsNewPHL (4) and MmNewPHL (5). Flavin adenine dinucleotide (FAD, 1), riboflavin (RF, 2) and flavin mononucleotide (FMN, 3) were used as references. (B) Fluorescence spectrum of MmNewPHL after in vitro oxidation with only FADox bound to the protein. The emission spectrum was recorded with a 420 nm excitation wavelength and the excitation spectrum was recorded with a 520 nm emission wavelength.