Short communication

The interaction of some water-soluble porphyrins and metalloporphyrins with human serum albumin

The interaction between human serum albumin (HSA) and two porphyrin-based tetra-cationic photosensitizers were investigated via spectroscopic techniques (steady-state, time-resolved fluorescence and circular dichroism) combined to theoretical calculations (molecular docking). Similarities and differences in the binding behavior of a porphyrin bearing peripheral chloro(2,2′bipyridine)platinum(II) complexes (4-PtTPyP) versus methyl groups (4-MeTPyP) attached in the N-pyridyl atoms were evaluated. There is a ground state association between HSA-porphyrins (static fluorescence quenching mechanism), with K_a values for 4-PtTPyP slightly higher than 4-MeTPyP. While the binding 4-MeTPyP is primarily enthalpically driven, 4-PtTPyP binds mainly due to hydrophobic forces in an entropically driven association. Far-UV CD showed that 4-PtTPyP perturbs the thermal stability of the protein, probably due to high volume of the porphyrin. Molecular docking suggested that 4-PtTPyP stablish a larger number of interactions with the neighboring amino acid residues. The likely location for the binding pocket is in subdomain IB, located in an external region of the protein structure. The bulky peripheral platinum(II)-complexes does not have a deleterious effect upon serum albumin binding spontaneity and extension, although modifies the nature of the complex formed.

The thermodynamic of the binding of N,N′,N″,N‴-tetramethyltetra-2,3-pyridinoporphyrazinatocopper(II) ([Cu(2,3-tmtppa)]⁴⁺), N,N′,N″,N‴-tetramethyltetra-3,4-pyridinoporphyrazinatocopper(II) ([Cu(3,4-tmtppa)]⁴⁺), N,N′,N″,N‴-tetramethyltetra-3,4-pyridinoporphyrazinatocobalt(II) ([Co(3,4-tmtppa)]⁴⁺), and N,N′,N″,N‴-tetramethyltetra-3,4-pyridinoporphyrazinatozinc(II) ([Zn(3,4-tmtppa)]⁴⁺), complexes to human serum albumin (HSA) has been studied. The binding constants (K) were obtained by analysis of optical absorption spectra of mentioned complexes at various HSA concentrations using SQUAD software. The values of K were obtained 9.77×10⁴ M⁻¹ for [Cu(2,3-tmtppa)]⁴⁺ and, 1.35×10⁵, 6.92×10⁵ and 2.00×10⁵ M⁻¹ for [Cu(3,4-tmtppa)]⁴⁺, [Zn(3,4-mtppa)]⁴⁺, and [Co(3,4-tmtppa)]⁴⁺, respectively, at 27 °C. The higher affinity of Cu-3,4-isomer towards HSA with respect to the 2,3-isomer can be attributed to favorable positioning of the cationic charges, which enables superior interaction with HSA. The thermodynamic parameters were calculated by van't Hoff equation. The enthalpy and entropy changes were 42.15 kJ mol⁻¹ and 236.02 J mol⁻¹ K⁻¹ for [Cu(2,3-tmtppa)]⁴⁺, 35.56 kJ mol⁻¹ and 216.75 J mol⁻¹ K⁻¹ for [Cu(3,4-tmtppa)]⁴⁺, 32.60 kJ mol⁻¹, 270.97 J mol⁻¹ K⁻¹ for [Zn(3,4-tmtppa)^]4+ and 47.75, 210.15 J mol⁻¹ K⁻¹ for [Co(3,4-tmtppa)]⁴⁺, respectively. The results indicate that the process is entropy driven suggesting that hydrophobic interactions are the main driving forces for complex formation. Binding of porphyrazine complexes quenches fluorescence emission of HSA. The quenching process obeys linear Stern–Volmer relationship. The values of Stern–Volmer constants (K_SV) and quenching rate constants (k_q) were determined by Stern–Volmer relationship. The values of K_SV and k_q were determined nearly 10⁶ M⁻¹ and 10¹⁵ M⁻¹ S⁻¹. Fluorescence studies also indicate that porphyrazine is probably bound to site I of HSA placed in sub-domain IIA, where tryptophan 214 is located. A competitive reaction by using phenyl putazone, a well-known site I marker, confirms this suggestion. The presence of hydrophobic cavity in the IIA sub-domain suggests that the interaction between HSA and porphyrazine is predominantly hydrophobic.

Non-covalent interactions between polymethine dyes of various types (cationic and anionic thiacarbocyanines as well as anionic oxonols and tetracyanopolymethines) and human serum albumin (HSA) were studied by means of absorption, fluorescence and circular dichroism (CD) spectroscopies. Complexation with the protein leads to a red shift of the dye absorption spectra and, in most cases, to a growth of the fluorescence quantum yield (Φ_f; for oxonols this growth is very small). The binding constants (K) obtained from changing the absorption spectra and Φ_f vary from 10⁴ to (5–6)×10⁷ M⁻¹. K for the anionic dyes is much higher than for the cationic dyes (the highest K was found for oxonols). Interaction of meso-substituted anionic thiacarbocyanines with HSA results in cis→trans isomerization and, as a consequence, an appearance and a steep rise of dye fluorescence. Binding to HSA gives rise to dye CD signals and in many cases is accompanied by aggregation of the dyes. These aggregates often exhibit biphasic CD spectra. The aggregates formed by the dyes alone are decomposed in the presence of HSA.

The high resolution structure of hemalbumin was determined by single crystal X-ray diffraction to a resolution of 1.9 Å. The structure revealed the protoporphyrin IX bound to a single site within a hydrophobic cavity in subdomain IB, one of the principal binding sites for long chain fatty acid. The iron is penta coordinated with the fifth ligand comprised of the hydroxyl oxygen of Tyr-161 (phenolic oxygen to heme plane distance: 2.73 Å) in an otherwise completely hydrophobic pocket. The heme propionic acid residues form salt bridges with His-142 and Lys-190, which together with a series of hydrophobic interactions, enclose and secure the heme within the IB helical motif. A detailed discussion of the structure together with its implications for the development of potential blood substitutes is presented.

The interaction of meso-tetrakis(p-sulfonatophenyl)porphyrin (TSPP) sodium salt to human serum albumin and β-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 R₀ ≈56 Å, between tryptophan and bound porphyrin, was estimated using the long distance Förster-type energy transfer formalism.

The binding of Mg-mesoporphyrin and mesoporphyrin to the primary binding site of human serum albumin (HSA) has been studied in the absence and in the presence of 1,2-dimyristoyl-sn-glycero-3- phosphatidylcholine/1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol (DMPC/DMPG) liposomes. The equilibrium constants of Mg-mesoporphyrin IX (MgMP) and mesoporphyrin IX (MP) for association to HSA were 1.7 · 10⁷ (M⁻¹) and 2.5 · 10⁷ (M⁻¹), respectively. The association constants for binding to the liposomes were: 1.5 · 10⁴ (M⁻¹) for MgMP, and 3.2 · 10⁴ (M⁻¹) for MP. The smaller value for the association constants of the MgMP relative to MP in both processes are interpreted as the effect of an out of plane position of Mg²⁺ and possible ligand co-ordination. HSA added to the liposomes with incorporated porphyrins results in the redistribution of bound molecules. For the MgMP this distribution can be interpreted by the competition of two independent binding processes, while the binding kinetics of MP significantly deviates from this model. The difference is explained by supposing a specific interaction between HSA and the liposomes in case of the free-base MP.