Modulation and Salt-Induced Reverse Modulation of the Excited-State Proton-Transfer Process of Lysozymized Pyranine: The Contrasting Scenario of the Ground-State Acid–Base Equilibrium of the Photoacid

Here we report on the excited-state behavior in terms of the excited-state proton-transfer (ESPT) reaction as well as the ground-state acid–base property of pyranine [8-hydroxypyrene-1,3,6-trisulfonate (HPTS)] in the presence of an enzymatic protein, human lysozyme (LYZ). HPTS forms a 1:1 ground-state complex with LYZ having the binding constant KBH = (1.4 ± 0.05) × 104 M–1, and its acid–base equilibrium gets shifted toward the deprotonated conjugate base (RO–), resulting in a downward shift in pKa. This suggests that the conjugate base (RO–) is thermodynamically more favored over the protonated (ROH) species inside the lysozyme matrix, resulting in an increased population of the deprotonated form. However, for the release of the proton from the excited photoacid, interestingly, the rate of proton transfer gets slowed down due to the “slow” acceptor biological water molecules present in the immediate vicinity of the fluorophore binding region inside the protein. The observed ESPT time constants, ∼140 and ...