Biophysical characterization of the interaction of human albumin with an anionic porphyrin

Abstract

The manuscript describes the characterization of the interaction between meso-tetrakis(p-sulfonatophenyl)porphyrin (TSPP) and human serum albumin (HSA). TSPP is a candidate for the photosensitization of structural and functional changes in proteins while HSA provides both an excellent protein model and binding and functional characteristics that could be explored in future applications of the approach. A combination of optical spectroscopic techniques (e.g., fluorescence spectroscopy, fluorescence lifetime, circular dichroism, etc.) and computational docking simulations were applied to better characterize the TSPP/HSA interaction. Recent advances have revealed that the complex formed by TSPP and HSA has become potentially relevant to biomedical applications, biomaterials research and protein photosensitized engineering. The study has determined a likely location of the binding site that places TSPP at a site that overlaps partially with the low affinity site of ibuprofen and places one of the [Formula: see text] groups of the ligand in proximity of the Trp214 residue in HSA. The characterization will enable future studies aimed at photosensitizing non-native functions of HSA for biomedical and biomaterial applications.

Keywords: Albumin; Docking; Porphyrin; Spectroscopy.

Graphical abstract

Fig. 1

Benesi-Hildebrand plot of TSPP binding affinity to HSA (▲).

Fig. 2

(A) Absorption of TSPP as a function of increasing concentration of HSA/ibuprofen complex. (B) Absorption of TSPP as a function of increasing concentration of HSA/warfarin complex. (C) Absorption of TSPP as a function of increasing concentration of HSA/hemin complex. In each figure the arrow indicates the direction of the spectral changes with increasing protein concentration. The initial and final concentrations are also indicated next to the arrow.

Fig. 3

(A) Temperature dependent Stern-Volmer plots of HSA fluorescence quenched by TSPP at 22 °C (△, —), 30 °C (•, ― ―), and 40 °C (▽, ‑ ‑).(B) Fluorescence quenching by porphyrin in the absence and presence of competing ibuprofen. Quenching of HSA by TSPP (▲,—); quenching of HSA/ibuprofen complex by TSPP (△,― ―).The solid lines represent the linear regression of the individual plots.

Fig. 4

Fluorescence decay of HSA in the absence of TSPP (black line) and in the presence of 3 μM TSPP (gray line).

Fig. 5

CD spectra of free (solid lines) and HSA-docked (dotted lines) PPIX (gray) and TSPP (black) in the region of the Soret band.

Fig. 6

(A) Docking simulation results for PPIX in the central cleft of HSA. The figure shows the remaining proximity with the Arg114 residue already participating in the binding to the heme pocket. (B) Docking simulation results for TSPP near the ibuprofen binding site. The figure shows the proximity of the 4 negatively charged sulfonic groups of the porphyrin with the positively charged Lys and Arg residues. (C) Overall view of HSA with docked PPIX (blue) and TSPP (red) and the crystal structure heme (yellow) at the sites shown in part A and B. The figure shows the position of each porphyrin relative to the lone Trp214 residue.