Excited-state proton transfer in hydroxynaphthaldehydes covalently bound to proteins

The kinetics and equilibrium of excited-state proton transfer (ESPT) in 2-h ydroxynaphthaldehyde-1 (HNA-2.1) bound to proteins (bovine serum albumin, c ytochrome c, and lysozyme) by an alkylamino bond was studied by means of fl uorimetric steady-state and time-resolved methods. The results were compare d to analogous data for l-hydroxy naphthaldehyde-4 (HNA-1.4) bound to prote ins and for other 2-naphthol derivatives bound to proteins by a sulfonamide bond. Conclusions concerning the influence on ESPT of the mode of binding and of intramolecular hydrogen bonds occurring in the case of HNA-2.1 were drawn. An intramolecular hydrogen bond enhances the rate of ESPT but the mo lecular environment in the protein leads to an, opposite effect by increasi ng reorganization energy during proton transfer. The results obtained prove that the mode of binding and the kind of group linking fluorophores to pro teins influence considerably the rate and mechanism of ESPT. In naphthol gr oups bound to proteins by an alkylamino bond, proton dissociation depends s trongly on the molecular environment in the macromolecule. This is due to t he short length of the alkylamino bridge and its small interaction with ele ctronic orbitals of the aromatic system. Fluorophores bound to proteins by a sulfonamide bond show a higher rate of ESPT, which is due partly to the e lectron withdrawing effect of the linking arm. The efficiency of ESPT for n aphthol groups bound to proteins by a sulfonamide bond is, in most cases, s ufficient for acidification of the medium and influence of the proton gradi ent in biological membranes.