SUPPORTING INFORMATION
Insight into the interaction of Inhaled Corticosteroids (ICS) with human serum albumin: a spectroscopic-based study.
Carlotta Pontremoli
a, Nadia Barbero
b, Guido Viscardi
b, Sonja Visentin
a*
a Molecular Biotechnology and Health Sciences Department, University of Torino, via Quarello 15, 10135
Torino, Italy. sonja.visentin@unito.it
b Department of Chemistry and NIS Interdepartmental Centre, University of Torino, via Pietro Giuria 7, 10125 Torino, Italy
1. UV-Vis spectroscopy
The absorption spectra of the protein at room temperature in absence and in presence of different concentrations of drugs betamethasone, prednisolone, triamcinolone= 2.0, 4.0, 6.0, 8.0, 10.0 μM;
flunisolide= 5.0, 10.0, 15.0, 20.0, 25.0 μM were recorded and shown in Fig. S1.
Fig. S1 UV–Vis spectra of HSA–drugs interaction (T = 296 K). [HSA]: black line. A) UV–Vis spectra of HSA–
betamethasone. B) UV–Vis spectra of HSA–flunisolide. C) UV–Vis spectra of HSA–prednisolone. (D) UV–Vis spectra of HSA–triamcinolone.
2. Binding parameters
By double logarithm regression curve, it is possible to obtain the number of binding sites (n). The plots obtained at room temperature using the following equation (eq. 3 in the paper1) are shown in Fig. S2. The slope of the line is the n value.
F (¿¿ 0−F)
F =log K
A+ n log[Q]
log ¿
-4,4 -4,2 -4,0 -3,8 -3,6 -3,4 -3,2
-1,4 -1,2 -1,0 -0,8 -0,6 -0,4 -0,2 0,0 0,2 0,4 0,6 0,8
1,0 HSA- Betamethasone HSA- Flunisolide HSA- Prednisolone HSA- Triamcinolone
Log [(F0-F)/F]
Log [Drug]
Fig. S2 Double logarithm regression curve of HSA-drugs, performed at 296 K.
3. Site Marker Competitive Binding Experiments
In order to investigate the site binding of corticosteroids, warfarin or ibuprofen were gradually added to the solution of HSA-corticosteroids complex ([HSA]= 5 μM; [corticosteroids]= 50 μM) and then fluorescence intensity was recorded. Fluorescence spectra were recorded in the range of 300-500 nm upon excitation at 280 nm; spectra are reported in Fig. S3 (competitive binding experiments with warfarin) and S5 (competitive binding experiments with ibuprofen). The excitation and emission slits were respectively 3 nm and 3 nm.
1 K.H. Ulrich, Molecular aspects of ligand binding to serum albumin, Pharmacol. Rev. 33 (1981) 17-53.
a) b)
c) d)
1
7
1
7 1
7 1
7
a) b)
c) d)
1 7
1 7
1 7
1 7
Fig. S3
Effect of warfarin on the quenching of HSA-corticosteroids complexes fluorescence: (a) HSA-betamethasone complex, (b) HSA-flunisolide complex, (c) HSA-prednisolone complex, (d) HSA-triamcinolone complex. Black line HSA- drug complex: ([HSA] = 5 μM [corticosteroids] = 50 μM; from 1 to 7 [warfarin] = 100, 200, 300, 400, 500, 750, and 1000 μM.
Fig. S4 Effect of ibuprofen on the quenching of HSA-corticosteroids complexes fluorescence: (a) HSA-betamethasone complex, (b) HSA-flunisolide complex, (c) HSA-prednisolone complex, (d) HSA-triamcinolone complex. Black line HSA- drug complex: ([HSA] = 5 μM [corticosteroids] = 50 μM; from 1 to 7 [ibuprofen] = 100, 200, 300, 400, 500, 750, and 1000 μM.
4. Thermodynamic parameters
The model of interaction between drug and protein could be obtained according to the data of enthalpy (ΔH) and entropy change (ΔS)2: (1) ΔH > 0 and ΔS > 0, hydrophobic forces; (2) ΔH < 0 and ΔS < 0, van der Waals interactions and hydrogen bonds; (3) ΔH < 0 and ΔS > 0, electrostatic interactions.
The values of ΔH and ΔS obtained by Van’t Hoff equations for the binding sites are shown in Table S1.
Table S1 Thermodynamic parameters for HSA- drugs complexes at different temperatures.
ΔH (kJ mol-1) ΔG (kJ mol-1) ΔS (J mol-1 K-1) Betamethasone
296 K -54,575.72 -19,062.59 -119.98
303 K -18,222.76
310 K -17,382.92
Flunisolide
296 K -68,161.45 -20,783.54 -160.06
303 K -19,633.11
310 K -18,542.69
Prednisolone
296 K -47,824.43 -20,260.53 -93.12
303 K -19,608.68
310 K -18,956.83
Triamcinolone
296 K -27,794.04 -17,020.91 151.40
303 K -18,080.74
310 K -19,140.58
2 Y.N. Ni, G.L. Liu, S. Kokot, Fluorescence spectrometric study on the interactions of isoprocarb and sodium 2- isopropylphenate with bovine serum albumin, Talanta, 76 (2008) 513-521.
5. Energy transfer
FRET (Fluorescence Resonance Energy Transfer) is a simple method to measure the distance between the acceptor (ligand) and the donor (tryptophan residues in the protein). The overlaps of the emission spectra of the protein HSA 5 μM (λ excitation at 280 nm; the excitation and emission slits were respectively 6 nm and 10 nm) and the absorption spectra of drugs (betamethasone, prednisolone, triamcinolone= 10.0 μM;
flunisolide= 25.0 μM) were performed at room temperature (296 K) and shown in Fig. S5.
Fig. S5 The overlaps of emission spectra of HSA and absorption spectra of betamethasone (A), flunisolide (B), prednisolone (C) and triamcinolone (D) at 296 K.
