Fluorescence solvatochromism of lumichrome (LC) was studied by steady-state and time-resolved fluorescence spectroscopy. The excited-state properties of LC do not show any correlation with solvent polarity, however, reasonably good correlation with solvent E(T)(30) parameter was observed. A quantitative estimation of contribution from different solvatochromic parameters, like solvent polarizability (π*), hydrogen bond donor (α), and hydrogen bond acceptor (β) ability of the solvent, was made using linear free energy relationship on the basis of Kamlet-Taft equation. The analysis reveals that hydrogen bond donating ability (acidity) of the solvent is the most important parameter that characterizes the excited-state behavior of lumichrome. Quantum mechanical calculations using density functional theory (DFT) were done to study the most stable structure and excited-state tautomerization process of LC toward the formation of isoalloxazines. Charge localization in the excited state and formation of hydrogen-bonded cluster through solvent hydrogen bond donation on the N10 atom of alloxazine moiety were predicted to be the key step toward this water-catalyzed tautomerization process.

The photophysical behavior of luminol (LH(2)) was studied in a variety of biologically relevant systems ranging from surfactants, cyclodextrin, and proteins using steady-state and time-resolved fluorescence spectroscopy. It was shown that, out of two possible LH(2) conformers present in solution, the sequestration of relatively less polar structure into the hydrophobic domain of biological media is the primary reason for decrease in fluorescence intensity. The efficacy of LH(2) fluorescence quenching is substantially higher in micellar subdomain of cationic surfactant and depends on the nature of the headgroup. The thermodynamic parameters like enthalpy (ΔH) and entropy (ΔS) change, etc., corresponding to the binding of LH(2) in the model water-soluble protein, bovine serum albumin (BSA), were estimated by performing the fluorescence titration experiment at different temperatures. The involvement of subdomain IA and IIA of BSA in LH(2) binding was confirmed from the ligand replacement process with bilirubin (BIL). The difference in ligand binding with structurally homologous human serum albumin (HSA) is discussed in terms of positive cooperativity among these two binding domains of BSA with a Hill coefficient (n(H)) value of 2.26 ± 0.18 and a half-maximal concentration (K(0.5)) of 5.74 ± 0.23 μM at 298 K.

The effect of solvent on the photoluminescence behavior of luminol was studied by steady-state fluorescence spectroscopy. The fluorescence spectral behavior of luminol is markedly different in polar protic solvents compared to that in aprotic solvents. A quantitative estimation of the contribution from different solvatochromic parameters, like solvent polarizibility (pi*), hydrogen-bond donor (alpha), and hydrogen-bond acceptor (beta), was made using the linear free energy relationship based on the Kamlet-Taft equation. The analysis reveals that the hydrogen-bond-donating ability (acidity) of the solvent is the most important parameter to characterize the excited-state behavior of luminol. Quantum mechanical calculations using density functional theory (DFT) predict the most stable structure, out of several possible tautomeric conformers of luminol with varying degrees of hydration. In the excited state, charge localization at specific points of the luminol phthalhydrazide moiety causes the solvent to interact primarily through hydrogen-bond donation.

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