Figure 4.

NMR spectra and site-specific binding isotherms for glycocholic acid binding to human I-BABP. (A) Two-dimensional gradient-enhanced HCACO spectrum of a sample containing a 3:1 mole ratio of [1′,2′-¹³C]glycocholate to unenriched I-BABP. The resonances corresponding to the unbound bile acid and the bile acid bound to sites 1 and 2 are designated U, 1, and 2, respectively. (B) Gradient- and sensitivity-enhanced ¹H/¹⁵N heteronuclear sequential quantum correlation (HSQC) spectrum (contour plot) of ¹⁵N labeled glycocholate for a sample containing a 3:1 mole ratio of [¹⁵N]glycocholate to I-BABP. (C) A stacked plot representation of the spectrum in B. The small peak at ¹H = 3.5 ppm and ¹⁵N = 123 ppm resulted from chemical exchange of ligand between site 2 and the unbound pool. (D) Site-specific NMR isotherms for the binding of [¹⁵N]glycocholate to I-BABP. The spectra at each mole ratio were recorded in triplicate by using separate samples for each mole ratio. The average ±95% confidence limits are reported for the unbound (squares), site 1 (triangles), and site 2 (open circles) resonances. The scaled peak volumes for site 2 include the measured volumes of peak 2 plus the volumes for the small exchange cross peak as seen in C.