Human Serum Albumin Affinity for Putrescine Using ITC and STD-NMR

Abstract

Understanding the binding interactions between protein-bound uremic toxins (PBUTs) and human serum albumin (HSA) is critical for advancing treatments for chronic kidney disease (CKD). While previous studies have suggested that putrescine, a diamine PBUT, exhibits moderate binding affinity to HSA, this study provides evidence of the contrary. Using isothermal titration calorimetry and saturation transfer difference nuclear magnetic resonance , we demonstrate that putrescine's interaction with HSA is weak, non-specific, and thermodynamically negligible in the range of conditions studied. Unlike earlier studies relying on spectroscopy techniques such as UV-visible absorption and fluorescence, which may overestimate binding strength, the results presented here highlight the limitations of indirect methodologies and underscore the importance of more sensitive approaches for accurate energy characterization. Our findings suggest that putrescine only weakly interacts non-specifically with HSA and may bind more preferentially to other plasma proteins, contributing to its accumulation in CKD patients.

Keywords: STD NMR; human serum albumin; isothermal titration calorimetry; protein-bound uremic toxins; putrescine.

Figure 1

1D-¹H NMR of putrescine (A) with chemical shifts ~1.5–2 ppm (H5, H6, H7, H8) and ~2.9–3.1 (H3, H4, H9, H10) [13] and STD-NMR spectra of putrescine–HSA (B). The large shift in the middle is for the reference solution (10% H₂O present).

Figure 2

ITC baseline-corrected thermograms (upper panel) and the fitting of multiple binding sites (lower panel). Putrescine with an initial concentration of 1.5 mM was injected to 0.1 mM HSA at 298 K and pH = 7.40. Baseline correction was conducted by subtracting the thermogram of putrescine injections into the buffer to account for the dilution heat.