Figure 3
From: Ligand entry in human ileal bile acid-binding protein is mediated by histidine protonation
Stopped-flow kinetic analysis of the pH-dependence of bile salt binding to human I-BABP. (A,B) Representative stopped-flow traces showing fluorescence changes upon mixing human I-BABP with GCA in buffer containing 20 mM potassium phosphate, 135 mM KCl, 10 mM NaCl, and 0.05% NaN3 at (A) pH = 7.2 and (B) pH = 5.8 at 15 °C (λex = 290 nm, λem > 320 nm). Final protein concentration was 1 µM. Final bile salt concentrations were 20, 30, 40, 50, 100, 150, 300 µM. The curve through the data points corresponds to a least-squares fit using a double exponential with observed rates depicted in (C). (C,D) Observed rates for the binding of (C) GCA and (D) GCDA to human I-BABP as a function of ligand concentration as determined from the stopped-flow experiments performed at pH = 5.8 (orange) and 7.2 (green). The solid lines are nonlinear least-squares fits according to the four-step sequential binding mechanism defined in eq. 3 with rate constants listed in Table 2.
