Sequential injection affinity chromatography utilizing an albumin immobilized monolithic column to study drug-protein interactions.
In this study, sequential injection affinity chromatography was used for drug-protein interactions studies. The analytical system used consisted of a sequential injection analysis (SIA) manifold directly connected with convective interaction media (CIM) monolithic epoxy disks modified by ligand-immobilization of protein. A non-steroidal, anti-inflammatory drug, naproxen (NAP) and bovine serum albumin (BSA) were selected as model drug and protein, respectively. The SIA system was used for sampling, introduction and propulsion of drug towards to the monolithic column. Association equilibrium constants, binding capacity at various temperatures and thermodynamic parameters (free energy DeltaG, enthalpy DeltaH) of the binding reaction of naproxen are calculated by using frontal analysis mathematics. The variation of incubation time and its effect in on-line binding mode was also studied. The results indicated that naproxen had an association equilibrium constant of 2.90 x 10(6)M(-1) at pH 7.4 and 39 degrees C for a single binding site. The associated change in enthalpy (DeltaH) was -27.36 kcal mol(-1) and the change in entropy (DeltaS) was -73 cal mol(-1)K(-1) for a single type of binding sites. The location of the binding region was examined by competitive binding experiments using a biphosphonate drug, alendronate (ALD), as a competitor agent. It was found that the two drugs occupy the same class of binding sites on BSA. All measurements were performed with fluorescence (lambda(ext)=230 nm, lambda(em)=350 nm) and spectrophotometric detection (lambda=280 nm).
Zacharis
Constantinos K
CK
Laboratory of Analytical Chemistry, Department of Chemistry, Aristotle University Thessaloniki, 541 24 Thessaloniki, Greece.
Kalaitzantonakis
Eftichios A
EA
Podgornik
Ales
A
Theodoridis
Georgios
G
D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity / Q000295:instrumentation* / Q000379:methods*; D009288:Naproxen / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D015203:Reproducibility of Results; D012710:Serum Albumin, Bovine / Q000737:chemistry* / Q000378:metabolism
D012710:Serum Albumin, Bovine; D009288:Naproxen