Comparative Analysis of KP-HSA Complex by Spectroscopic Methods

Abstract

The main objective of the presented study was to characterize the high (HAS) and low anity (LAS) binding sites of ketoprofen (KP) in human serum albumin (HSA) structure with the use of spectrouorescence and proton nuclear magnetic resonance spectroscopy. In vitro uorescence analysis was used to estimate the eect of KP on the HSA uorescence. The association constants Ka [M -1 ] of KP-HSA complex in the HAS were determined with the use of Scatchard, Klotz, and Hill analysis. The quenching KQ [M -1 ] constants were determined on the basis of the SternVolmer equation. Binding of ketoprofen to plasma protein was also studied with the use of 8-anilinonapthalene-1-sulfonic acid (ANS) and 5-dimethylaminonaphthalene-1-sulfonic acid (DNSA) as the uorescence probes in IIIA and IIA subdomains of HSA, respectively. To estimate the cooperativeness in proteins Hill's coecient nH was used. The analysis of proton nuclear magnetic resonance spectra of KP in the presence of HSA allows us to observe the interactions between aromatic rings of the drug and the rings of amino acids located in the hydrophobic subdomains of the protein on the basis of the changes of chemical shifts [ppm] of drug protons resonances. Moreover the Ka constants [M -1 ] of KP-HSA complex in the LAS were determined.