Two-dimensional correlation method applied to Yb3+ vibronic sideband spectroscopy: Discrimination of fluorescence spectral features arising from different Yb3+ binding sites

Ytterbium (Yb3+) vibronic sideband (VSB) spectroscopy allows the identification of the lanthanide ligands both in model compounds and in Yb3+-reconstituted metalloproteins. In some cases there exist multiple binding sites which complicate the Yb3+ fluorescence spectrum. The presence of different sites can be deduced from the variation of the intensity of the VBSs, when the wavelength of excitation is varied. A general two-dimensional (2D) correlation method developed by Noda can be potentially used to discriminate the different Yb3+ binding sites. A very simple procedure has been further developed so that, by using a few different spectra, usable information can be extracted from the 2D spectrum; the spectra were obtained with different wavelengths of excitation. This method is applied successfully in the present work on Yb3+-reconstituted transferrin, which contains two spectroscopically different Yb3+ sites, in order to determine any structural difference between the two sites. (C) 1995 John Wiley & Sons, Inc.