Excited-state proton transfer as a biological probe. Determination of rate constants by means of nanosecond fluorometry

Abstract

Ionization constants of organic acids in the excited state may differ by several orders of magnitude from those observed in the ground state (Weller, A. (1961), Progr. React. Kinet. 1, 187). Nanosecond time-resolved fluorescence spectroscopy has been used to detect proton transfer in the excited state. It is shown that fluorescence lifetime data can be used to obtain the rate constants for excited-state proton transfer of 2-naphthol in aqueous solution. Three distinct methods are described for the calculation of rate constants, and good agree-D ifferences in the chemical and physical properties of molecules in their excited states relative to their ground states can be used as the basis for designing probes of biological microenvironments. Organic acids exhibit excited-state ionization constants which differ by several orders of magnitude from those observed in the ground state (Forster, 1950a;Weller, 1961;Wehry and Rogers, 1966; Vander Donckt, 1970). Weller (1952), as well as other workers, has used steadystate fluorescence methods to calculate the forward and reverse rate constants for proton transfer. Rate constants for proton transfer depend on solvent environment and on the character of proton donors and acceptors. A quantitative measure of the environment of the probe can be made by calculating rates of proton transfer. These rates are expected to be sensitive to changes in protein conformation.Quantitation of rate constants using kinetic methods yields the same values as those obtained by steady-state techniques using model compounds such as 2-naphthol. However, more information about the reacting system can be obtained by analysis of fluorescence decay curves, and such analysis can be applied to fluorescent probes attached to proteins. In this approach, light absorption and fluorescence are used as a relaxation technique to study reactions on a time scale of sec or less. This type of relaxation technique differs from the traditional relaxation techniques in the extent of the perturbation. In excited-state studies the equilibrium is shifted by converting the reaction partners to new species (the excited state) rather than by changing intensive parameters such as the temperature or pressure.Previous work (DeLuca et al., 1971 ;Bowie et al., 1972) has demonstrated the potential of excited-state proton transfer for investigating the active sites of enzymes. The aim of the present ment is obtained between the values for rate constants ob. tained by these methods and those obtained with the steadystate approach. Rapid rates of excited-state proton transfer are found with 2,6-naphtholsulfonate in aqueous solution. When this chromophore is adsorbed to bovine serum albumin, little or no proton transfer is observed. The potential application of excited-state proton transfer to studies of biological macromolecules is discussed.paper is to show how rate constants for excited-state proton transfer can be obtained from fluorescence lifetime measurements and to indicate the advantage of this approach over steady-state m...

Nuclear Magnetic Resonance Spectroscopy Investigations of Naphthalene-Based 1,2,3-Triazole Systems for Anion Sensing

“…Deprotonation occurs during the excited state lifetime of the molecule. This phenomenon correlates to the widely studied photophysics of 2-Naphthol [25,[67][68][69][70][71] where the molecule undergoes the ESPT process at high pH conditions, revealing emission of the Naptholate anion around 410 nm [67]. Observation of the emissions from both the deprotonated NpTP and NpTP-F − hinted at the fact that the excited state reaction is partially completed during the excited state lifetime [67].…”

“…For ion recognition, complicated molecules with multistep syntheses are used to create highly conjugated systems with common scaffolds such as ureas, amides, and/or phenolic groups [19,20]. Photoinduced electron transfer (PET) [21,22], metal-to-ligand charge transfer (MLCT) [22,23]; excimer/exciplex formation [23], intramolecular charge transfer (ICT) [24]; and excited state intra/intermolecular proton transfer (ESPT) [25,26] are some of the signaling mechanisms by which the anions are detected.…”

Excited-State Reactions

“…[1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] In the case of 2-naphthol, the pKa decreases from 9.5 in the ground state to 2.8 in the excited state. 18 In acid solution, the emission is from naphthol, with an emission maximum of 357 nm ( Figure 18.1).…”

“…Such spectra are shown in Figure 18.7 for 2,6-naphtholsulfonate, a sulfonated form of 2-naphthol. In order to obtain TRES within the time resolution available at that time, 14 the reaction was slowed by the addition of ethanol. The TRES shift to longer wavelength following the excitation pulse.…”

Excited-state proton transfer from pyranine to acetate in methanol

“…To analyse the observed decay characteristics of HPTS in the presence of acetate, we now use a simple kinetic model 14,15 as follows In this case, the excited photoacid HA* donates its proton directly to the acetate ion to produce A -*. k′ 1 and k′ 2 are the pseudo-first order rate constants respectively for the forward and backward processes in the excited state.…”