Experimental and computational characterization of photosensitized conformational effects mediated by protoporphyrin ligands on human serum albumin

Abstract

When investigating the interaction between proteins and protoporphyrins in aqueous solution, one typically has to contend with the tendency of the latter to form polydispersed aggregates. The interference of aggregated protoporphyrins manifests, at least, at two levels: aggregates sequester the majority of the protoporphyrin molecules in solution and prevent their interaction with the proteins, but also their presence interferes with optical experiments such as absorption and fluorescence spectroscopy. In this study we present a protocol which uses dialysis and centrifugation to eliminate the aggregates and yield solutions dominated by non-covalent complexes of albumin (HSA) and protoporphyrins. The elimination of the aggregates enabled us to observe effects which had not been previously observed such as eliminating the discrepancy between the binding constants obtained through the quenching of HSA fluorescence and the one obtained through the emission of the protoporphyrins. Moreover the elimination of the aggregated protoporphyrins enabled us to reveal the occurrence of fluorescence resonance energy transfer (FRET) between the Trp214 residue of HSA and the porphyrin ligands. FRET data were then used to estimate the location of metal free as well as Zn-protoporphyrin IX relative to the well-known location of Trp214. This information was used to refine docking simulations to find the best binding site for the two protoporphyrins. In addition we observed that the irradiation of the protoporphyrins in the visible region prompts small conformational changes in HSA that appear to be largely due to tertiary modifications.

Multi-wavelength analytical ultracentrifugation of human serum albumin complexed with porphyrin

“…Tin as well as Zinc-protoporphyrin IX (Sn(IV)PPIX and Zn(II)PPIX), were purchased from Frontier Scientific Inc. (Logan, UT). A detailed description of the sample preparation can be found elsewhere [14]. Briefly, a small amount of solid protoporphyrin was initially dissolved in 1 ml of DMSO and allowed to equilibrate overnight to yield a highly concentrated solution of monomeric porphyrin.…”

“…Briefly, a small amount of solid protoporphyrin was initially dissolved in 1 ml of DMSO and allowed to equilibrate overnight to yield a highly concentrated solution of monomeric porphyrin. The concentration of this stock was then determined spectroscopically upon dilution in DMSO using the Beer-Lambert equation [14]. An aliquot (100–200 μl) of this stock solution was added to 10 ml of a solution containing ~10 μM HSA and allowed to equilibrate for an hour at room temperature.…”

“…The dialyzed solutions were collected and centrifuged for 5 minutes at 11.5 g, using an EPPENDORF MiniSpin Plus centrifuge (Hauppauge, NY). After centrifugation the supernatant of the porphyrin/protein mixtures contains only apo-HSA and the HSA/protoporphyrin complex and the rest of the aggregates have been eliminated [14]. At this point the remaining supernatant was pooled and then separated into four 2 ml aliquots.…”

“…Observing the OD of the 278 nm peak (0.5 OD), HSA was combined with solution 1 to increase the OD at 278 nm up to 0.75 OD for solution 2 and 1.0 OD for solution 3, while roughly maintaining the OD of the Soret band. The concentration of the protein in each sample was determined spectroscopically using the value of the absorption of the solutions at 278 nm and assuming a molar extinction coefficient ε 278nm = 3.4 × 10 4 M −1 cm −1 for HSA [14] and ε 280nm = 3.281 × 10 4 M −1 cm −1 [17, 18].…”

“…One challenge is created by the tendency of the protoporphyrin to form polydispersed aggregates which do not interact with the protein and interfere with other analytical techniques (e.g., fluorescence spectroscopy). We have shown that this challenge can be overcome by a multistep sample preparation that leaves in solution only the complexes between the monomeric porphyrins and apo-HSA [14]. A second challenge arising from the poor solubility of the porphyrins is the inability to determine their molar extinction coefficient in aqueous solutions, and to determine the fraction of complexed HSA.…”