Results:                                2011                        2012                       2013                       2014                       2015                        2016



Bioligand – HSA interaction, characterized by fluorescence spectroscopy

a) The interaction of tolmetin (TOL), with human serum albumin (HSA) in physiological buffer solution (pH 7.40) was studied by fluorescence and UV-vis absorption spectroscopy at different temperatures, combined with time resolved fluorescence measurements. The experimental results showed that there was a strong fluorescence quenching of HSA by tolmetin. Using the continuous variation method, a single class of binding sites for TOL on HSA was put in evidence. The binding constants Ka were calculated using a nonlinear fit to the experimental data. The thermodynamic parameters of the tolmetin interaction with HSA were measured according to the van’t Hoff equation. The enthalpy change (ΔH0) and the entropy change (ΔS0) were calculated to be - 21.74 kJ/mol and 23.75 J/mol K, respectively, which indicated that the interaction between TOL and HSA was driven mainly by van der Waals forces. Based on time - resolved fluorescence, we concluded that a dynamic quenching mechanism between TOL and HAS is present. Moreover, evidence for efficient Förster resonance energy transfer is presented, from which we infer that the binding site of TOL is proximal (r = 2.35 nm) to the Trp-214, in subdomain IIA of HSA.

Analisis of ligand binding to HSA using NMR relaxation rates measurements

a) Ligand binding studies are commonly analysed using the simplest two-site model in which the ligand  L can bind to any of the n identical independent binding sites on a protein P. In this case , the observed relaxation rate of a proton belonging to the ligand ,Roba, is the population weighted average of the free (Rf), and bound (Rb) ligand relaxation rate: Robs = (1-χb) Rf + χbRb. Defining  the total ligand concentration as CL = [Lf] + [Lb]  and nCP =[Lb] + [P] , together with the definition of the dissociation constant Kd = [Lf][P]/[Lb],the bound population can be calculated as being χ =- α – (α2 – β)1/2 and α = (CL + nCP +  Kd)/2Cand β = nCP/CL. Following the χ dependence on CL, while keeping CP constant, n, Kd and Rb can be calculated using a non-linear regression analysis. For the beginning we will check this model for the ibuprofen/HSA system.

We have investigated the bioligand – human serum albumin interaction by measuring the longitudinal relaxation rate R1,obs of some protons belonging to ligand as a function of its concentration. The concentration of the human serum albumin was kept constant at 0.2 mM. The bioligand concentration varied  between 4 – 60 mM.We have investigated the binding properties of ibuprofen, 5 – fluorouracil, cytarabine and paracetamol.. The NMR relaxation experiments were performed with a 500 MHz  Bruker NMR spectrometer, using the well known inversion recovery pulse sequence. The obtained results are:










From the obtained results can be concluded that cytarabine has the highest affinity for HAS while paracetamol the lowest.



Text Box: Bioligand – macromolecule intermolecular interactions studied by spectroscopic and calorimetric techniques  
Text Box: National  Institute for R&D of Isotopic and Molecular Technologies Cluj-Napoca


R1b (sec)-1


Kd (mM)

ibuprofen / HSA

3.56 ± 0.04

36 ± 1

32.5 ± 1

5-fluorouracil / HSA

1.531 ± 0.34

39 ± 8

18.8 ± 2

paracetamol / HSA

1.756 ± 0.03

46 ± 1

34.8 ± 1

cytarabine / HSA

0.523 ± 0.002

28 ± 1

2.9 ± 0.5