Fluorescence assays of G6PD variants

Intrinsic fluorescence and 8-anilino-1-naphthalenesulfonic acid (ANS) assays of the recombinant human WT-G6PD and G6PD variants were conducted as described formerly [7 (link),21 (link)]. Both assays were monitored in a Perkin-Elmer LS-55 spectrofluorometer (Wellesley, MA, USA). Emission fluorescence spectra from 310 to 500 nm were recorded after excitation at 295 nm, whereas the ANS assays were conducted using an excitation wavelength of 395 nm and recording the emission fluorescence spectra from 400–600 nm. All the intrinsic fluorescence and ANS assays were conducted as described previously [7 (link),21 (link),25 (link)]. Background fluorescence from the buffer (blank), and buffer plus ANS was subtracted from those that contained the respective protein.