A modified multisite Stern-Volmer equation for the determination of conditional stability constants and ligand concentrations of soil fulvic acid with metal ions.

In this work, we modify the multisite Stern-Volmer (MSV) equation for fitting fluorescence titration curves. Under the condition of a static quenching mechanism, the MSV postulates an underlying 1:1 fulvic acid (FA)/copper coordination ratio at multisites. Approximates of six fitting parameters characterize the stability constants (K1 and K2) of FA ligands with Cu2+, micromolar ligand site concentrations (CL1 and CL2), the unquenched, steady-state fractional fluorescence contributions (fx), and the residual fluorescence intensity (IRES). Prior to its application to actual FA titration data, the MSV function is simulated, and its predictive ability is confirmed by titrating a mixture of model fluorophores, glycyl-L-tryptophan and L-tryptophan with Cu2+ at pH 6. Molecular fluorescence measurements of FA are acquired at a fixed spectral position (lambda(ex) = 335 nm; lambda(em) = 450 nm), and FA is titrated with copper in triplicate at three pH values-5, 6, and 7. An objective analysis of log K(1) and K(2) values supports several site organization schemes, including (i) subtle, cooperative interaction, (ii) interfering molecular conformations, and (iii) aggregate forms. Site densities (CL1 and CL2) are consistent across varied pH. The f(1) is indicative of a pH-induced spectral shift of a fluorophore and convincingly associates with a transect in the Deltalambda = 25 synchronous fluorescence spectrum and with the preexponential terms describing the time-dependent fluorescence decay. The MSV and its parent one-site version are equivalent for data fitting but are only simple approximations of a FA ligand system with more complex molecular fundamentals.