Chromatographic analysis of the effects of fatty acids and glycation on binding by probes for Sudlow sites I and II to human serum albumin

Abstract

The primary endogenous ligands of human serum albumin (HSA) are non-esterified fatty acids, with 0.1–2 moles of fatty acids normally being bound to HSA. In type II diabetes, fatty acid levels in serum are often elevated, and the presence of high glucose results in an increase in the non-enzymatic glycation of HSA. High-performance affinity chromatography (HPAC) was used to examine the combined effects of glycation and the presence of long chain fatty acids on the binding of HSA with R-warfarin and L-tryptophan (i.e., probes for Sudlow sites I and II, the major sites for drugs on this protein). Zonal elution competition studies were used to examine the interactions of myristic acid, palmitic acid and stearic acid with these probes on HSA. It was found that all these fatty acids had direct competition with R-warfarin at Sudlow site I of normal HSA and glycated HSA, with the glycated HSA typically having stronger binding for the fatty acids at this site. At Sudlow site II, direct competition was observed for all the fatty acids with L-tryptophan when using normal HSA, while glycated HSA gave no competition or positive allosteric interactions between these fatty acids and L-tryptophan. These data indicated that glycation can alter the interactions of drugs and fatty acids at specific binding sites on HSA. The results of this study should lead to a better understanding of how these interactions may change during diabetes and demonstrate how HPAC can be used to examine drug/solute-protein interactions in complex systems.

Rapid Investigation and Screening of Bioactive Components in Simo Decoction via LC-Q-TOF-MS and UF-HPLC-MD Methods

“…In site II, the docking scores of polymethoxy flavonoids were higher than at site I. This phenomenon was consistent with the tendency of site I to bind bulky heterocyclic anionic compounds and site II to aromatic carboxylates [ 57 ]. Moreover, the effect of a hydroxyl on glycosyl was found to be less than that of a hydroxyl on the parent nucleus, perhaps because of the large area of steric hindrance [ 58 ].…”

“…Working factors of pH and temperature influenced the activity of HSA, time of incubation influenced the binding degree of binders, eluting steps removed the disturbance of unbound compounds, and dissolution reagent was necessary for the dissociation of HSA-drug complexes. Optimum conditions in the study were established by referring to related studies [ 56 , 57 , 58 ] which performed single factor experiments in previous work. This study was performed at 37 °C (physiological temperature) with pH 7.4 (the plasma condition) to provide optimal reaction conditions.…”

“…In order to expound how bioactive drugs conjugate with HSA, a molecular docking simulation was used for further illustration in the active sites and binding degree of the ligands on HSA. The interactions of drugs with HSA typically occur at two major hydrophobic sites, known as Sudlow’s site I (subdomain IIA) and site II (subdomain IIIA), which are located in subdomains [ 57 ]. Many studies have verified that warfarin and ibuprofen were specific binders for site I and site II, respectively.…”

Glycation of human serum albumin impairs binding to the glucagon-like peptide-1 analogue liraglutide

“…The two most common drug binding sites of albumin are located in the hydrophobic cavities of subdomain IIA and IIIA, known as Sudlow's sites I and II, respectively (53). Non-esterified medium-and long-chain fatty acids, such as myristic and palmitic acid, can bind to seven sites on HSA, three of which are located on Sudlow's drug binding sites I and II (54,55). In particular, the albumin region from residues 377-582, including part of domain II and the entire domain III was found to be the primary binding site for long chain fatty acids (56) and, more precisely, the key roles of R410 (Sudlow's site II) and K525 (domain IIIB) for fatty acid binding has been reported (57).…”

Serum Levels and Removal by Haemodialysis and Haemodiafiltration of Tryptophan-Derived Uremic Toxins in ESKD Patients

“…Thus, heparin provokes the release of lipolytic enzymes [44], leading to hydrolysis of triglycerides and release of free fatty acid and increasing their plasma concentrations. It has been shown that free fatty acids displace drugs and ligands (also for IS, IAA and Trp) from their protein binding sites [45,46] and that Trp increases displacement of IS and IAA [47]. IS, IAA and Trp primarily bind to Sudlow site II on albumin, with IS having the highest affinity [47][48][49][50].…”