One-step purification of PhKAT and spectrophotometric analysis of the cofactor (PLP) binding to apo-KAT.
(A) SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Lanes ∼1–10, verification of the purity of PhKAT in each elution fraction from the metal-affinity column. Protein fractions in lanes ∼2–10 were used for further analysis and crystallization. The positions of the protein standards (molecular masses, 97, 66, 45, 30, 20.1, and 14.4 kDa) are indicated. (B) UV–visible absorption spectra of as-isolated PhKAT (lower spectrum) and its cofactor-binding form (holo-PhKAT, upper spectrum). The lower spectrum was obtained with the as-purified protein, and the upper spectrum was recorded after mixing 20 µM PhKAT with 20 µM PLP. The spectrum shows a maximum at 361 nm. (C) Spectrophotometric titration of PLP for 10 and 20 µM PhKAT. (D) Relationship between the equilibrium-binding response and the concentrations of PLP and PhKAT. The solid lines represent the fitting curve obtained by the 2-site binding model with hill slopes using Prism5 software. (E) Summary table of binding parameters from D. Errors are the S.E. from the fit of the data. The binding affinity of a second binding site for PLP was stronger than a first binding site in PhKAT (Kd1>Kd2).