Near-UV Circular Dichroism and Second-Derivative Fluorescence Spectroscopy as Complementary Tools for Studying Ligand–Albumin Interactions

Spectroscopic techniques offer significant potential for investigating ligand–protein interactions, particularly for assessing conformational modifications and binding affinity. In the present study, a complementary approach combining near-UV circular dichroism (CD) and second-derivative fluorescence spectroscopy was applied to evaluate how two representative nonsteroidal anti-inflammatory drugs—phenylbutazone (PHB, a marker of Sudlow’s site I) and ketoprofen (KP, a marker of Sudlow’s site II)—influence the tertiary structure of human serum albumin in its native form (HSA) and after glycation by glucose (gHSAGLC), fructose (gHSAFRC), and glucose–fructose syrup (gHSAsyrup). The results demonstrate that glycation substantially modifies the tertiary structure of HSA and decreases its drug-binding capacity at Sudlow’s sites I and II, with the most pronounced conformational changes observed for gHSAFRC, confirming fructose as the most reactive glycation agent. PHB induced distinct conformational rearrangements, including a characteristic increase in ellipticity near ~290 nm, indicating perturbations in the chiral microenvironment surrounding Trp214 within Sudlow’s site I. By contrast, KP induced weaker, site-specific structural changes, primarily within Phe-rich hydrophobic domains of site II. Glycation consistently increased the polarity and solvent exposure of aromatic residue microenvironments—particularly within Tyr-rich regions—while the local environment of Trp214 remained comparatively stable. These findings suggest that PHB and KP modulate the conformational flexibility of glycated HSA predominantly by reorganizing Tyr-rich regions rather than directly perturbing Trp214. Overall, the study shows that glycation heterogeneity significantly influences protein–drug interactions, with important implications for altered pharmacokinetics in diabetes and metabolic disorders. The combined application of near-UV CD and second-derivative fluorescence spectroscopy offers a sensitive and complementary strategy for distinguishing structural differences between non-glycated and glycated HSA and for characterizing drug–albumin interactions at the tertiary structural level of the macromolecule.