Comparative Analysis of KP-HSA Complex by Spectroscopic Methods
M. Mąciażek-Jurczyk, J. Równicka-Zubik, R. Dyja and A. Sułkowska
Department of Physical Pharmacy, Medical University of Silesia, Jagiellońska 4, 41-200 Sosnowiec, Poland
Received: August 9, 2012; in final form: February 2, 2013
Full Text PDF
The main objective of the presented study was to characterize the high (HAS) and low affinity (LAS) binding sites of ketoprofen (KP) in human serum albumin (HSA) structure with the use of spectrofluorescence and proton nuclear magnetic resonance spectroscopy. In vitro fluorescence analysis was used to estimate the effect of KP on the HSA fluorescence. 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 Stern-Volmer equation. Binding of ketoprofen to plasma protein was also studied with the use of 8-anilino-napthalene-1-sulfonic acid (ANS) and 5-dimethyl-amino-naphthalene-1-sulfonic acid (DNSA) as the fluorescence probes in IIIA and IIA subdomains of HSA, respectively. To estimate the cooperativeness in proteins Hill's coefficient 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 Δ σ [ppm] of drug protons resonances. Moreover the Ka constants [M-1] of KP-HSA complex in the LAS were determined.

DOI: 10.12693/APhysPolA.123.673
PACS numbers: 33.50.-j, 33.50.Dq, 82.56.-b, 87.14.-g, 87.14.E-