The pH-dependent conformational transition of β-lactoglobulin modulates the binding of protoporphyrin IX

We have investigated the interaction between PPIX and β-lactoglobulin (β-lg) as a function of the pH of the solution. β-lg is a small globular protein (MW ≈18 kDa) with a very well characterized structure that reveals several possible binding sites for ligands. The interaction with β-lg affects the photophysical properties of PPIX. The shift of PPIX emission maximum, excitation maximum and the increase of the fluorescence intensity is an indicator that binding between the porphyrin and β-lg occurs. The binding constant appears to be modulated by the pH of the solution. Spectroscopic measurements do not reveal any significant energy transfer between the Trp residues of β-lg and PPIX, however, fluorescence anisotropy decay measurements confirm the binding and the modulation introduced by the pH of the solution. Since β-lg has been shown to be stable within the range of pH adopted in our experiments (5.0–9.0), the results suggest that PPIX binds a site affected by the pH of the solution. Because of the crystallographic evidence an obvious site is near the aperture of the interior β-barrel however an alternative (or concurrent) binding site may still be present.