Interaction of Aggregated Cationic Porphyrins with Human Serum Albumin

The interaction of an equilibrium mixture of monomeric and aggregated cationic trans-5,15-bis(N-methylpyridinium-4-yl)-10,15-bis-diphenylporphine (t-H2Pagg) chloride salt with human serum albumin (HSA) has been investigated through UV/Vis absorption, fluorescence emission, circular dichroism and resonant light scattering techniques. The spectroscopic evidence reveals that both the monomeric t-H2Pagg and its aggregates bind instantaneously to HSA, leading to the formation of a tight adduct in which the porphyrin is encapsulated within the protein scaffold (S430) and to clusters of aggregated porphyrins in electrostatic interaction with the charged biomolecules. These latter species eventually interconvert into the final S430 species following pseudo-first-order kinetics. Molecular docking simulations have been performed to get some insights into the nature of the final adduct. Analogously to hemin bound to HSA, the obtained model supports favorable interactions of the porphyrin in the same 1B subdomain of the protein. Hydrophobic and van der Waals energy terms are the main contributions to the calculated ΔGbind value of −117.24 kcal/mol.


Introduction
Porphyrinoids are an important class of both synthetic and naturally occurring compounds. They possess electronic structures that are responsible for intense absorption bands in the visible region of the spectrum, together with fluorescence emission [1]. Their structural features are enriched by metal ions inserted into the macrocyclic core that bring coordinating properties and redox activity. For these reasons, porphyrins are involved in many biologically relevant molecules where they play important roles in electron transfer (cytochromes), oxygen transport (hemoglobin) and light harvesting (chlorophylls) [2]. Their peculiar optical properties are deeply influenced by the specific microenvironment, making them useful spectroscopic probes [3][4][5][6]. Their propensity to interact with biological systems has also fostered their use as sensitizers for singlet oxygen production upon light irradiation. This ability is at the basis of photodynamic therapy (PDT), where these compounds are actively investigated for producing reactive oxygen species (ROS) able to exert an efficient anticancer activity directly in vivo [7].
Porphyrin aggregation is another relevant phenomenon that stems from an interplay of hydrophobic or solvophobic effects together with electrostatics, hydrogen bonding and other specific molecular recognitions [8]. The formation of self-organized or selfassembled supramolecular systems is well documented in the literature and strongly affects the spectroscopic properties [9] and the reactivity [10] with respect to the isolated monomeric species. Furthermore, when porphyrins self-assemble onto chiral templates, Int. J. Mol. Sci. 2023, 24,2099 3 of 13 porphyrin-protein system, evaluating the chiroptical and photophysical properties exhibited by the chromophores in the presence of HSA. Scheme 1. Pictorial sketch of the molecular structure of cationic t-H2Pagg (chloride salt) and a fractal aggregate formed by increasing the ionic strength of the solution.

Results and Discussion
As well as t-CuPagg, upon salt addition t-H2Pagg self-aggregates in aqueous solution forming large fractal clusters [41], although the amount of salt to obtain the total aggregation of this chromophore is much higher than that necessary for the metal derivative [42].
The extinction spectrum of the neat t-H2Pagg porphyrin in phosphate buffer 1 mM pH = 7.4 is shown in Figure 1 (black curve). In line with its monomeric character, it displays a rather sharp Soret band centered at 419 nm together with four weaker Q-bands at a lower energy.

Results and Discussion
As well as t-CuPagg, upon salt addition t-H 2 Pagg self-aggregates in aqueous solution forming large fractal clusters [41], although the amount of salt to obtain the total aggregation of this chromophore is much higher than that necessary for the metal derivative [42].
The extinction spectrum of the neat t-H 2 Pagg porphyrin in phosphate buffer 1 mM pH = 7.4 is shown in Figure 1 (black curve). In line with its monomeric character, it displays a rather sharp Soret band centered at 419 nm together with four weaker Q-bands at a lower energy. Scheme 1. Pictorial sketch of the molecular structure of cationic t-H2Pagg (chloride salt) and a fractal aggregate formed by increasing the ionic strength of the solution.

Results and Discussion
As well as t-CuPagg, upon salt addition t-H2Pagg self-aggregates in aqueous solution forming large fractal clusters [41], although the amount of salt to obtain the total aggregation of this chromophore is much higher than that necessary for the metal derivative [42].
The extinction spectrum of the neat t-H2Pagg porphyrin in phosphate buffer 1 mM pH = 7.4 is shown in Figure 1 (black curve). In line with its monomeric character, it displays a rather sharp Soret band centered at 419 nm together with four weaker Q-bands at a lower energy. UV/Vis spectra of monomeric t-H2Pagg no salt added (black curve), and t-H2Pagg aggregates (red curve) obtained upon addition of NaCl. Inset: kinetic traces recorded for the decrease of monomeric t-H2Pagg at λ = 419 nm (black circles) and the formation of t-H2Pagg aggregates at λ = 450 nm (red circles). Solid lines represent the result of the global fit obtained with The addition of NaCl (100 mM) promotes the gradual decrease of the B-band and the simultaneous formation of a new band at 450 nm, related to the self-aggregation of porphyrin into fractal assemblies (red curve). The extinction spectra clearly show that this ionic strength value leads to a partial aggregation with an almost equal distribution between monomer and aggregated porphyrin. This experimental finding is in line with previous results and the kinetic profiles displayed in the inset of Figure 1 show a short incubation period, followed by an autocatalytic growth, in agreement with the formation of diffusion limited clusters (DLA), and preceded by a reaction-limited activated step [43].
When a rather high concentration of protein (100 µM) is added to the solution, the extinction spectrum displays an immediate change: the B-band at 419 nm related to the monomeric porphyrin undergoes a bathochromic shift to 430 nm ( Figure 2, light green curve), while the spectrum displays an off-set from the baseline probably due to light scattering. This spectrum reveals also that still an almost equal amount of this new species (S 430 ) is present together with the porphyrin clusters. During 1 h, the gradual conversion of the band relative to the aggregate into the band at 430 nm occurs, which increases in intensity (dark green curve). This evolution is also reported on the inset of Figure 2, where the intensity at 430 nm recorded over time was fit with Equation (2). The value of n obtained from the fit (close to 1) indicates a typical exponential behavior. netic profiles displayed in the inset of Figure 1 show a short incubation perio autocatalytic growth, in agreement with the formation of diffusion limited clu preceded by a reaction-limited activated step [43].
When a rather high concentration of protein (100 μM) is added to the so tion spectrum displays an immediate change: the B-band at 419 nm related t porphyrin undergoes a bathochromic shift to 430 nm ( Figure 2, light green spectrum displays an off-set from the baseline probably due to light scatterin reveals also that still an almost equal amount of this new species (S430) is pres the porphyrin clusters. During 1 h, the gradual conversion of the band relativ into the band at 430 nm occurs, which increases in intensity (dark green curv is also reported on the inset of Figure 2, where the intensity at 430 nm record fit with Equation (2). The value of n obtained from the fit (close to 1) indicat nential behavior. The resonant light scattering (RLS) technique confirms similar feature tinction ( Figure 3). The formation of large supramolecular structures in solut dition is emphasized by the appearance of an intense signal with an app around 470 nm, at the red edge of the absorption band of the clusters ( Fig  Upon protein addition, the RLS spectrum shows an instantaneous increase o sity, followed by a steady reduction. The time-dependence of this signal is rep of Figure 3, and it shows a similar exponential behavior to that observed extinction spectra.
Additionally, CD spectroscopy gives important information about the s Monomeric t-H2Pagg, as well as its aggregated form, do not display any CD dition of protein causes an instantaneous appearance of an exciton-split ICD negative Cotton effect in the absorption region of the clusters (light green c that porphyrin aggregates are responsible for this spectral feature. Upon protein addition, the RLS spectrum shows an instantaneous increase of the signal intensity, followed by a steady reduction. The time-dependence of this signal is reported in the inset of Figure 3, and it shows a similar exponential behavior to that observed previously in the extinction spectra. Additionally, CD spectroscopy gives important information about the system ( Figure 4). Monomeric t-H 2 Pagg, as well as its aggregated form, do not display any CD signals. The addition of protein causes an instantaneous appearance of an exciton-split ICD signal showing negative Cotton effect in the absorption region of the clusters (light green curve), indicating that porphyrin aggregates are responsible for this spectral feature.  These kinds of ICD bands are similar, even if much less intense, to those observed when the t-CuPagg clusters interact with the α-helices of poly(D-glutamic acid) [42], or for t-H2Pagg interacting on the surface of M13 bacteriophages [36]. Following over time the spectral evolution, a continuous transformation of the spectrum can be detected, that changes in shape and intensity, leading to a weaker monosignate signal having a negative Cotton effect (dark green curve). These ICD bands are in agreement with the monomeric nature of the porphyrin interacting with the protein [32].   These kinds of ICD bands are similar, even if much less intense, to those observed when the t-CuPagg clusters interact with the α-helices of poly(D-glutamic acid) [42], or for t-H2Pagg interacting on the surface of M13 bacteriophages [36]. Following over time the spectral evolution, a continuous transformation of the spectrum can be detected, that changes in shape and intensity, leading to a weaker monosignate signal having a negative Cotton effect (dark green curve). These ICD bands are in agreement with the monomeric nature of the porphyrin interacting with the protein [32]. These kinds of ICD bands are similar, even if much less intense, to those observed when the t-CuPagg clusters interact with the α-helices of poly(D-glutamic acid) [42], or for t-H 2 Pagg interacting on the surface of M13 bacteriophages [36]. Following over time the spectral evolution, a continuous transformation of the spectrum can be detected, that changes in shape and intensity, leading to a weaker monosignate signal having a negative Cotton effect (dark green curve). These ICD bands are in agreement with the monomeric nature of the porphyrin interacting with the protein [32].
As previously anticipated, fluorescence emission experiments were performed in order to obtain further information. Figure 5 shows the usual fluorescence emission spectrum of monomeric t-H 2 Pagg, characterized by the typical two bands pattern for the aqueous porphyrin solution at 666 and 714 nm (black curve). As previously anticipated, fluorescence emission experiments were performed in order to obtain further information. Figure 5 shows the usual fluorescence emission spectrum of monomeric t-H2Pagg, characterized by the typical two bands pattern for the aqueous porphyrin solution at 666 and 714 nm (black curve). As reported in the literature, on increasing the ionic strength, aggregate formation leads to a substantial fluorescence quenching and a slight shift of the maxima, now centered at 673 and 715 nm (red curve) [36].
The addition of HSA causes immediately a relevant modification of the spectrum profile, which becomes much more asymmetric displaying a consistent intensity increase (about five-fold) and a blue shift (≈10 nm) of the main band now centered at 663 nm (light green curve), together with a modest enhancement and a red shift of the band at a longer wavelength (≈9 nm). Both these features slightly increase in intensity over time (dark green curve).
The formation of an adduct between porphyrin and protein is also remarked by the moderate increase of emission quantum yield value of this species ( = 0.072) with respect to that of the free base t-H2Pagg, registered in bulk solution ( = 0.06), similar to that reported for the interaction of the same porphyrin with the M13 bacteriophage [36]. This result underlines the assumption of the exclusion of water molecules from the solvation shell of the chromophore as a consequence of the binding to HSA, therefore resulting in effective dielectric changes of the microenvironment around the porphyrins. Thus, it is possible to hypothesize that after the initial electrostatic binding, porphyrins are confined into an internal hydrophobic site of the protein.
This model is also confirmed by the marked difference between the reported static fluorescence anisotropy values for monomeric free porphyrin ( = 0.017) and the porphyrin-protein adduct ( = 0.059). These values suggest a higher rotational freedom for the monomeric species with respect to the adduct form, in line with the binding between the fluorophore and the larger protein [44].
The experimental spectroscopic evidence suggests that the addition of protein on a solution containing a mixture of monomers and clusters shifts instantaneously the equi- As reported in the literature, on increasing the ionic strength, aggregate formation leads to a substantial fluorescence quenching and a slight shift of the maxima, now centered at 673 and 715 nm (red curve) [36].
The addition of HSA causes immediately a relevant modification of the spectrum profile, which becomes much more asymmetric displaying a consistent intensity increase (about five-fold) and a blue shift (≈10 nm) of the main band now centered at 663 nm (light green curve), together with a modest enhancement and a red shift of the band at a longer wavelength (≈9 nm). Both these features slightly increase in intensity over time (dark green curve).
The formation of an adduct between porphyrin and protein is also remarked by the moderate increase of emission quantum yield value of this species (φ F = 0.072) with respect to that of the free base t-H 2 Pagg, registered in bulk solution (φ F = 0.06), similar to that reported for the interaction of the same porphyrin with the M13 bacteriophage [36]. This result underlines the assumption of the exclusion of water molecules from the solvation shell of the chromophore as a consequence of the binding to HSA, therefore resulting in effective dielectric changes of the microenvironment around the porphyrins. Thus, it is possible to hypothesize that after the initial electrostatic binding, porphyrins are confined into an internal hydrophobic site of the protein.
This model is also confirmed by the marked difference between the reported static fluorescence anisotropy values for monomeric free porphyrin (r = 0.017) and the porphyrinprotein adduct (r = 0.059). These values suggest a higher rotational freedom for the monomeric species with respect to the adduct form, in line with the binding between the fluorophore and the larger protein [44].
The experimental spectroscopic evidence suggests that the addition of protein on a solution containing a mixture of monomers and clusters shifts instantaneously the equilibrium towards the formation of a new species, S 430 , together with the porphyrin aggregates strongly interacting with HSA. Chirality is immediately induced on the porphyrin assemblies as proved by the bisegnated ICD spectra. As mentioned above, under neutral conditions, the overall charge on the protein is close to −15. Considering that the nominal ratio [HSA]/[t-H 2 Pagg] is 20, a large excess of the protein is present for each porphyrin monomer. Additionally, strong electrostatic interactions between the positively charged porphyrin clusters and the negatively charged HSA should be expected leading to the observed ICD on the aggregates. At the same time, all the spectroscopic features analyzed seem to suggest a preferential interaction between HSA and the monomeric form of t-H 2 Pagg, which leads to the subsequent slow degradation of t-H 2 Pagg aggregates and the formation of an adduct between porphyrin and HSA (S 430 ), where the monomeric porphyrin experiences a different and chiral microenvironment (Scheme 2). librium towards the formation of a new species, S430, together with the porphyrin aggregates strongly interacting with HSA. Chirality is immediately induced on the porphyrin assemblies as proved by the bisegnated ICD spectra. As mentioned above, under neutral conditions, the overall charge on the protein is close to −15. Considering that the nominal ratio [HSA]/[t-H2Pagg] is 20, a large excess of the protein is present for each porphyrin monomer. Additionally, strong electrostatic interactions between the positively charged porphyrin clusters and the negatively charged HSA should be expected leading to the observed ICD on the aggregates. At the same time, all the spectroscopic features analyzed seem to suggest a preferential interaction between HSA and the monomeric form of t-H2Pagg, which leads to the subsequent slow degradation of t-H2Pagg aggregates and the formation of an adduct between porphyrin and HSA (S430), where the monomeric porphyrin experiences a different and chiral microenvironment (Scheme 2).

Scheme 2. Model for the interaction of aggregated (black clusters) and monomeric (red circles) t-H2Pagg and HSA (green).
Molecular Modeling. The interaction between t-H2Pagg and HSA was investigated at the molecular level by performing molecular docking simulation employing the crystal structure of HSA in complex with heme (PDB ID 1N5U). Considering that t-H2Pagg and heme share similar chemical scaffolds, the docking search was focused on the heme binding site which consist of a hydrophobic D-shaped cavity located in the 1B subdomain of HSA ( Figure 6A). The resulting poses were submitted to MM-GBSA calculations, as described in the Methods section, in order to identify the binding conformation with the lowest binding free energy (ΔGbind) which afforded a ΔGbind value of −117.24 kcal/mol.
As displayed in Figure 6B, the results revealed that t-H2Pagg might occupy the heme binding site on HSA by establishing π-stacking interactions with F134, Y161, F157 and H146. Moreover, several π-cation interactions were observed between (i) one of the pyrrole rings of t-H2Pagg and R117 and (ii) one of the N-methylpyridinium moieties and H146, F149 and F157. In addition, one of the pyrrole subunits might engage a H-bond with the hydroxy group of Y161, while electrostatic interactions might be established between E153 and one of the N-methylpyridinium rings. Finally, the binding of t-H2Pagg to HSA might be stabilized by hydrophobic contacts involving P118, M123, L135, A158, L154, I142 and L115, and van der Waals interactions with V122, A126, V116, F134, F165, L139, L185, S192 and S193.
The contribution of each energy component to the ΔGbind, which include electrostatic (ΔGColoumb), van der Waals (ΔGVdW), π-π packing (ΔGPacking), covalent (ΔGCovalent), H-bond (ΔGH-bond), self-contacts (ΔGSelfCont), hydrophobic (ΔGLipo) and the solvation free energy (ΔGSolvGB) [45] are reported in Table 1. According to the outcomes, the most favorable contributions to the ΔGbind derive from the van der Waals and hydrophobic energy terms which therefore represent the primary driving forces for the interaction between t-H2Pagg and HSA.

Scheme 2. Model for the interaction of aggregated (black clusters) and monomeric (red circles) t-H 2 Pagg and HSA (green).
Molecular Modeling. The interaction between t-H 2 Pagg and HSA was investigated at the molecular level by performing molecular docking simulation employing the crystal structure of HSA in complex with heme (PDB ID 1N5U). Considering that t-H 2 Pagg and heme share similar chemical scaffolds, the docking search was focused on the heme binding site which consist of a hydrophobic D-shaped cavity located in the 1B subdomain of HSA ( Figure 6A). The resulting poses were submitted to MM-GBSA calculations, as described in the Methods section, in order to identify the binding conformation with the lowest binding free energy (∆G bind ) which afforded a ∆G bind value of −117.24 kcal/mol.
As displayed in Figure 6B, the results revealed that t-H 2 Pagg might occupy the heme binding site on HSA by establishing π-stacking interactions with F134, Y161, F157 and H146. Moreover, several π-cation interactions were observed between (i) one of the pyrrole rings of t-H 2 Pagg and R117 and (ii) one of the N-methylpyridinium moieties and H146, F149 and F157. In addition, one of the pyrrole subunits might engage a H-bond with the hydroxy group of Y161, while electrostatic interactions might be established between E153 and one of the N-methylpyridinium rings. Finally, the binding of t-H 2 Pagg to HSA might be stabilized by hydrophobic contacts involving P118, M123, L135, A158, L154, I142 and L115, and van der Waals interactions with V122, A126, V116, F134, F165, L139, L185, S192 and S193.
The contribution of each energy component to the ∆G bind , which include electrostatic (∆G Coloumb ), van der Waals (∆G VdW ), π-π packing (∆G Packing ), covalent (∆G Covalent ), Hbond (∆G H-bond ), self-contacts (∆G SelfCont ), hydrophobic (∆G Lipo ) and the solvation free energy (∆G SolvGB ) [45] are reported in Table 1. According to the outcomes, the most favorable contributions to the ∆G bind derive from the van der Waals and hydrophobic energy terms which therefore represent the primary driving forces for the interaction between t-H 2 Pagg and HSA.

Materials
trans-5,15-bis(N-methylpyridinium-4-yl)-10,15-bis-diphenylporphine (t-H2Pagg) was purchased from Mid-Century Chemicals as the chloride salt and used as received. Stock solutions of porphyrin were prepared dissolving the solids in dust-free Millipore water and stored in the dark. Solution concentrations were determined from the known molar extinction coefficient at the Soret maximum (ε = 2.40 × 10 5 M −1 cm −1 ) [39]. Human serum albumin (HSA) was purchased from Sigma and stock solutions were prepared by dustfree Millipore water in phosphate buffer 0.01 M pH = 7.4. All experiments were carried out in dust-free Millipore water and in 1 mM phosphate buffer, pH = 7.4. All other reagents were supplied by Aldrich Chemicals Co. (St. Louis, MO, USA) and used without further purification.

Materials
trans-5,15-bis(N-methylpyridinium-4-yl)-10,15-bis-diphenylporphine (t-H 2 Pagg) was purchased from Mid-Century Chemicals as the chloride salt and used as received. Stock solutions of porphyrin were prepared dissolving the solids in dust-free Millipore water and stored in the dark. Solution concentrations were determined from the known molar extinction coefficient at the Soret maximum (ε = 2.40 × 10 5 M −1 cm −1 ) [39]. Human serum albumin (HSA) was purchased from Sigma and stock solutions were prepared by dust-free Millipore water in phosphate buffer 0.01 M pH = 7.4. All experiments were carried out in dust-free Millipore water and in 1 mM phosphate buffer, pH = 7.4. All other reagents were supplied by Aldrich Chemicals Co. (St. Louis, MO, USA) and used without further purification.

Methods
UV/Vis measurements were conducted on an Agilent 8453 diode array spectrophotometer. Kinetic experiments were followed in the thermostated compartment of the instrument, with a temperature accuracy of 0.1 K at 298 K. The analyses of the extinction kinetic profiles have been performed by a non-linear fit of the absorption data according to the following equations reported in the literature: (i) where Ext 0 , Ext ∞ , k 0 , k c , m and n are the parameters to be optimized and, (ii) where Ext 0 , Ext ∞ , k and n are the parameters to be optimized (Ext t , Ext 0 and Ext ∞ are the extinction at time t, at starting time and at the end of aggregation, respectively) [46]. Fluorescence emission and resonance light scattering (RLS) experiments were performed on a Jasco model FP-750 spectrofluorometer equipped with a Hamamatsu R928 photomultiplier, adopting for RLS experiments a synchronous scan protocol with a rightangle geometry [3]. Fluorescence emission was filtered with a high-pass filter (cutoff: 600 nm) to remove excitation overtones. RLS spectra were not corrected for the absorption of the samples. Fluorescence anisotropy measurements were obtained on the same instrument equipped with linear polarizers (Sterling Optics 105UV). Fluorescence anisotropy (r) is defined by the following equation: where I V H and I VV are the fluorescence intensities with horizontal and vertical polarization, respectively. The different transmission efficiency of polarized light by both excitation and emission monochromators has been accounted for by correcting the I V H value through the factor G = I HV /I HH that is a correction factor strictly dependent on monochromator wavelength and slit widths [47]. The fluorescence quantum yield (φ F ) was calculated on the basis of the following equation: where I F is the area under the fluorescence emission spectrum, A λex is the absorbance value at the excitation wavelength, S refers to the sample, whereas R refers to the reference fluorophore of known quantum yield (tetrakis(N-methylpyridinium-4-yl)porphyrin, φ F (R) = 0.047 in aqueous buffer solution) [36]. The circular dichroism (CD) spectra were recorded on a JASCO J-720 spectropolarimeter, equipped with a 450 W xenon lamp.

Molecular Modeling
Molecular docking simulation was performed by using the crystal structure of HSA in complex with heme (PDB ID 1N5U) [48]. The protein structure was prepared as described elsewhere [49]. The t-H 2 Pagg structure was built by means of Maestro (Schrödinger Release 2021-4: Maestro, Schrödinger, LLC, New York, NY, USA, 2021) and submitted to a conformational search by means of MacroModel (Schrödinger Release 2021-4: Macro-Model, Schrödinger, LLC, New York, NY, USA, 2021) applying the default settings. The charges and geometry of the lowest energy conformation were optimized by QM calculations at B3LYP-D3/LACVP** theory level using Jaguar [50]. The so obtained structure was docked into HSA binding site by means of the software PLANTS v 1.2 [51]. The binding site was set in order to contain all the residues within 10 Å from the co-crystallized ligand. The docking was performed setting speed 1 as accuracy level and ChemPLP as scoring function. The resulting docking poses were rescored by performing MM-GBSA calculation by means of Prime tool [52] employing the VSGB solvation model and "minimize" as sampling method. The minimization involved all the residues situated at 10 Å from the ligand. The pose characterized by the lowest binding free energy value was chosen for the analysis and representation. Ligand-protein interactions were analyzed by means of Discovery Studio Visualizer (BIOVIA, Dassault Systèmes, Discovery Studio Visualizer, v.20.1.0.19295, San Diego, CA, USA: Dassault Systèmes, 2019) and Maestro packages.

Concluding Remarks
Fractal aggregates of the cationic copper(II) derivative of t-H 2 Pagg are useful chiroptical probes for a variety of biomolecules. These micrometric sized species are positively charged and they readily bind to species bearing negative charges. Moreover, if the substrate is chiral, chirality is transferred to the porphyrin clusters, and usually amplified. In the present investigation, the parent metal-free porphyrin exhibits the same propensity with the additional benefit of being an emitting species in its monomeric form. The interaction of the porphyrin clusters with HSA is a biphasic process: (i) a fast, electrostatically driven contact occurs at the mixing, and chirality is induced on the porphyrin aggregates, and (ii) the fractal aggregates are slowly disrupted by the protein, leading to a quite stable adduct where the porphyrin is bound in the protein scaffold and protected by the solvent. This latter species has been modeled and the porphyrin share the same pocket of hemin in the 1B subdomain of the HSA. Our results show that even cationic porphyrins in aggregated form are able to interact efficiently with serum proteins. Further investigations are on the way to get more insights into the dynamics of this process.