Assessment of chemical exchange in tryptophan-albumin solution through (19)F multicomponent transverse relaxation dispersion analysis

Abstract

A number of NMR methods possess the capability of probing chemical exchange dynamics in solution. However, certain drawbacks limit the applications of these NMR approaches, particularly, to a complex system. Here, we propose a procedure that integrates the regularized nonnegative least squares (NNLS) analysis of multiexponential T2 relaxation into Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments to probe chemical exchange in a multicompartmental system. The proposed procedure was validated through analysis of (19)F T2 relaxation data of 6-fluoro-DL-tryptophan in a two-compartment solution with and without bovine serum albumin. Given the regularized NNLS analysis of a T2 relaxation curve acquired, for example, at the CPMG frequency υ CPMG = 125, the nature of two distinct peaks in the associated T2 distribution spectrum indicated 6-fluoro-DL-tryptophan either retaining the free state, with geometric mean */multiplicative standard deviation (MSD) = 1851.2 ms */1.51, or undergoing free/albumin-bound interconversion, with geometric mean */MSD = 236.8 ms */1.54, in the two-compartment system. Quantities of the individual tryptophan species were accurately reflected by the associated T2 peak areas, with an interconversion state-to-free state ratio of 0.45 ± 0.11. Furthermore, the CPMG relaxation dispersion analysis estimated the exchange rate between the free and albumin-bound states in this fluorinated tryptophan analog and the corresponding dissociation constant of the fluorinated tryptophan-albumin complex in the chemical-exchanging, two-compartment system.

Figure 1

Skematic of the two-compartmental 6F-Trp system. A 3-mm inner diameter tube made of semi-permeable dialysis membrane was inserted into a 5-mm NMR tube to separate the BSA-6F-Trp complex solution from the sole 6F-Trp solution for the ¹⁹F transverse relaxation experiments.

Figure 2

T₂ distributions resulting from the regularized NNLS analysis of ¹⁹F T₂ relaxation curves. The echo time present in the CPMG pulse sequence was τ_CPMG = 2 ms. A single T₂ component was depicted in (A) the 6F-Trp solution and (B) the BSA-6F-Trp complex solution, respectively, while (C) two T₂ components were observed in the two-compartmental 6F-Trp system. The χ² statistics for goodness-of-fit tests of the regularized NNLS-derived fits of T₂ relaxation curves exhibited χ² = 2317, p = 0.41 for the 6F-Trp solution (Figure S1A), χ² = 2370, p = 0.16 for the BSA-6F-Trp complex solution (Figure S1B), and χ² = 2206, p = 0.92 for the two-compartmental 6F-Trp system (Figure S1C), all with degrees of freedom = 2303.

Figure 3

¹⁹F effective transverse relaxation rates as a function of CPMG field strength for 6F-Trp compartments. Relaxation dispersion data collected in free/bound exchanging 6F-Trp are presented with the associated fitting curves. The fitting curves to relaxation dispersion data using Eq. 2 (slow exchange expression) are displayed in dash lines, while those using Eq. 3 (skewed population P_A > P_B approximation) are in solid lines. The relaxation dispersions are plotted in (A) $R_2^{\mathit{eff}}$ vs. CMPG frequency and (B) $R_2^{\mathit{eff}}$ vs. half of echo-spacing τ_CPMG.