Interaction of human serum albumin with novel 3,9-disubstituted perylenes

Abstract

Human serum albumin (HSA) has been used as a model for the binding of a number of different ligands, including polyaromatic hydrocarbons, to proteins. In this case we have investigated the interaction of HSA with a novel set of perylene derivatives. Di-substituted perylene analogues have been synthesized as potentially useful organic photovoltaic materials. Their photophysical properties may make them viable for fuel cell applications too. However, these molecules are poorly soluble especially in aqueous solvents. Binding to water-soluble proteins may provide a way to solubilize them. At the same time one can study whether the photophysical processes initiated by the irradiation of a perylene ligand can cause conformational changes to the host protein. With the present study we demonstrated that of the three perylene derivatives investigated only one, the dimethoxy analogue, has a significant affinity for HSA at a binding site near the bottom of the central cleft (in proximity of the Trp214 residue). The small affinity prevents any significant photoinduced changes to occur in the protein.

Fig. 1

Secondary structure of human serum albumin (PDB code: 109X). Domains: I (Blue), II (green), A and B subdomains are depicted in dark and light shades, respectively. Domain IIIA (yellow), IIIB (red). Tryptophan (orange) is located at Site-T which is indicated approximately by the region in the square. Marked are also the sites for ibuprofen (red) and warfarin (purple) (Color figure online)

Fig. 2

Computational model of the location of the DMOP (cpk color scheme) binding sites on HSA and the close up view of the binding configurations. The conformation of Phe 211 and Trp214 are also shown. This configuration represents the lowest energy among the clusters at this site as determined by the Lamarckian algorithm adopted with Autodock 4.0

Fig. 3

Stern–Volmer plots of the static quenching caused by DMOP on HSA fluorescence a S–V plots for HSA (filled square), HSA/ibuprofen complex (filled triangle) and HSA/warfarin complex (filled circle). b S–V plots produced by the addition of DOOP and PDO on free HSA. To a 5 μM solution of HSA at ~pH 7.4, increasing amounts of perylene were added. For each addition the absorption spectrum was recorded between 250 and 500 nm and the fluorescence spectrum of the protein was recorded in the 305–450 nm range upon excitation at 295 nm. The fluorescence intensity was calculated as the integral between 305 and 450 nm of the emission spectrum. The integral was then normalized by dividing its value for the optical density at 295 nm. The parameter so calculated yielded the values of F₀ and F represented in the pictures. In the case of HSA/warfarin or HSA/ibuprofen, the concentration of the competing ligand (ibuprofen or warfarin) was incubated for 1 h at a concentration of 10 μM (i.e., double the concentration of albumin)

Fig. 4

Fluorescence emission spectra of perylenes as a function of the concentration of HSA. a DMOP, b DOOP, c PDO. To μM solutions of each perylene, additional aliquots were added from a stock solution containing 30 μM of HSA. After each addition the spectra of the perylenes were recorded upon excitation at 422 nm

Fig. 5

Benesi–Hildebrand plot with linear regression for the addition of HSA to aqueous solutions containing DMOP (filled triangle), DOOP (filled square) and PDO (filled circle). These plots were derived from the spectra shown and described in Fig. 4. The spectra were first corrected for the instrumental response. The fluorescence intensity was retrieved from the integral of the spectra in the 450–600 nm range normalized by dividing the value of the integral by the optical density of the perylenes at the excitation wavelength (422 nm). ΔF was then calculated as the difference between the emission intensity in the absence of HSA and the fluorescence intensity in the presence of a concentration [HSA] of the protein

Fig. 6

Benesi–Hildebrand plot with linear regression obtained from the fluorescence of DMOP upon addition of: HSA (filled square), HSA/ibuprofen complex (filled triangle), HSA/warfarin complex (filled circle). The procedure to obtain the plot is identical to that explained in Fig. 5. In this case to the solution containing the perylene derivatives it was added a stock solution containing a 2:1 concentration ratio of ibuprofen (or warfarin) and albumin. The concentration of albumin in the stock was 30 μM

Fig. 7

CD Spectra for HSA/perylenes complexes. The plots show the overlay between the dichroic signal in the region of the amide absorption of the protein in the protein/perylene complex before irradiation of the ligand (blue) and after exposure to 56 W/cm² irradiation (red). It can be noted that irradiations does not change to the spectra of HSA. a HSA/DMOP complex, b HSA/DOOP complex, c HSA/PDO complex. Spectra were recorded from solutions containing a diluted amount of protein. The original solution containing 5 μM HSA and ~5 μM perylenes was diluted as to obtain an optical density of ~0.9 at 220 nm in 1 mm quartz cells. This is the optimal optical density for CD measurements. This implied a ~10 times dilution of the original solution (Color figure online)