Characterization of the interaction of metal-protoporphyrins photosensitizers with β- lactoglobulin

Irradiation of ZnPPIX Complexed with Bovine β-Lactoglobulin Causes Chemical Modifications and Conformational Changes of the Protein

“…Prior to laser irradiation, the spectrum of ZnPPIX:BLG shows a broad peak with a maximum at 394 nm in the Soret region of the porphyrin, as well as a peak at 278 nm in the UV (Figure 1A). The peak at 394 nm is consistent with a mixture of monomeric and aggregated ZnPPIX [43] with a larger fraction of the latter, which is not eliminated by our sample preparation under acidic conditions. One also notices two Q-bands at 556 nm and 590 nm, which represent a D 4h symmetry (essentially flat) for the porphyrin macrocycle [73].…”

“…A sub-nanosecond component (α 1 , τ 1 in Table 1A) and a component near 1.6-1.7 ns (α 2 , τ 2 in Table 1A). They combine to yield an average lifetime of ~1.40 ns, which is shorter than that of BLG alone, likely because of distance-dependent fluorescence resonance energy transfer (FRET) between the Trp residues and ZnPPIX ligand, which we reported previously [43].…”

“…Figure S1B shows the spectra of ZnPPIX in buffer and in the complex with BLG at the same pH. The spectrum of the complex is narrower and shifted by 4 nm to longer wavelengths, indicating that in the complex, ZnPPIX is mostly monomeric, while without the protein, it remains significantly aggregated [43,75]. The region > 500 nm shows two Q-bands that are significantly redshifted and narrower compared to the pH 5 samples and indicate that even at this pH and upon forming a complex with BLG, the porphyrin maintains a flat D 4h symmetry.…”

“…This is likely caused by the fact that, despite our sample preparation, smaller oligomers of the ZnPPIX are likely dominant in solution at acidic pH, whereas most PPIXs are much less soluble. These oligomers are unlikely to bind BLG [43,89,90] and prompt significant photochemical mechanisms [76]. As a result, the ZnPPIX:BLG complex is a nearly negligible fraction (<3%) of the sample.…”

“…Among our earlier investigations were the self-assembly of metal-free porphyrins (cationic and neutral) with BLG [39,40,42]. In recent studies, we have demonstrated that metal-free (mf ) and metal-PPIXs (including ZnPPIX) non-covalently bind BLG dimers, likely in the groove at the interface between the two monomers (Scheme S1) [43]. In the same study, we showed that the formation of protein-PPIX complexes is much more efficient at alkaline than acidic pH and concluded that the larger efficiency is not due to the Tanford transition of BLG, which occurs at a location distant from the binding site of the porphyrin but is instead due to the fact that the PPIXs remain much more aggregated at acidic pH; thus fewer porphyrin monomers are available to bind the protein.…”