Spectroscopic Studies on the Interaction of a Water Soluble Porphyrin and Two Drug Carrier Proteins

Abstract

The interaction of meso-tetrakis(p-sulfonatophenyl)porphyrin (TSPP) sodium salt to human serum albumin and beta-lactoglobulin was studied by steady-state and dynamic fluorescence at different pH of aqueous solutions. The formation of TSPP J-aggregates and a noncovalent TSPP-protein complex was monitored by fluorescence titrations, which depend on pH and on the protein nature and concentration. The complex between TSPP and protein displays a heterogeneous equilibrium with large changes in the binding strength versus pH. The large reduction of the effective binding constant from pH 2 to 7 suggests that electrostatic interactions are a major contribution to the binding of TSPP to the aforementioned proteins. TSPP aggregates and TSPP-protein complex exhibit circular dichroism induced by the presence of the protein. Circular dichroism spectra in the ultraviolet region show that the secondary structure of both proteins is not extensively affected by the TSPP presence. Protein-TSPP interaction was also examined by following the intrinsic fluorescence of the tryptophan residues of the proteins. Fluorescence quenching by acrylamide and TSPP itself also point to small changes on the protein tertiary structure and a critical distance R(0) approximately 56 A, between tryptophan and bound porphyrin, was estimated using the long distance Förster-type energy transfer formalism.

Translational and Rotational Motions of Albumin Sensed by a Non-Covalent Associated Porphyrin Under Physiological and Acidic Conditions: A Fluorescence Correlation Spectroscopy and Time Resolved Anisotropy Study

“…We can exclude contribution from TSPP to this signal since, taking into account the absence of chirality in TSPP molecules, as expected no CD signal was detected for TSPP samples without BSA in the UV-Vis range. Nevertheless, an intense induced CD arising from the chiral packing of the molecules into large aggregates was detected in the visible range [11]. At higher ratios, the signal is less negative.…”

“…Since the two albumins, HSA and BSA present closely homologous structures and have the same isoelectric point [27], we have taken into account the binding constants already determined for TSPP interaction with HSA, K b~5 × 10 6 M −1 and K b~1 ×10 6 M −1 respectively at pH=2 and pH= 7 [11], from which a fraction of bound TSPP of 85% at the former and 67% at the latter pH, could be withdrawn. These values match reasonably the amplitudes obtained for the long component and therefore, can be assigned to TSPP monomer/BSA complex where the porphyrin is in a hydrophobic environment of the protein matrix and hence "protected" from changes in the solution pH.…”

“…We have previously shown [11] that the anionic watersoluble porphyrin, meso-tetrakis (p-sulfonatophenyl) porphyrin sodium salt-TSPP, binds to albumins with relatively high binding constants, (K B~3 -5×10 6 M −1…”

“…Moreover, under acidic conditions TSPP is prevalently present in solution as a zwitterion. This has proved to contribute decisively to the formation of highly ordered molecular aggregates of the porphyrin at relatively low [TSPP] induced by different polymeric templates [12][13][14] including albumins [11,15]. These aggregates of J-type are particularly interesting in view of their possible applications in nonlinear optics, nanometer-sized photoconductors, and light harvesting systems and contributed also for the relevance of the studies carried on at pH=2.…”

Investigating Tryptophan Quenching of Fluorescein Fluorescence under Protolytic Equilibrium

“…The photophysical parameters of a fluorophore are obviously dependant on various external environmental factors such as pH, polarity, temperature, ion concentration, membrane potential, etc. 2,6,7 However, fluorophore emission after conjugation (covalent or otherwise) to a macromolecule can be completely different from the free probe, under the same environmental conditions, [8][9][10][11] due to the possibility that the emission properties may be affected by the microenvironment of the binding site, primarily, via non radiative mechanisms. 12-,,15 Of particular significance is the quenching interaction of specific amino acids in the protein, like tryptophan.…”