2-DIMENSIONAL CORRELATION METHOD APPLIED TO YB3-BAND SPECTROSCOPY - DISCRIMINATION OF FLUORESCENCE SPECTRAL FEATURES ARISING FROM DIFFERENTYB3+ BINDING-SITES( VIBRONIC SIDE)

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