Distinction of the two binding sites of serum transferrin by resonance Raman spectroscopy

Abstract

The resonance Raman (RR) data for a variety of transferrin samples were investigated to explore differences between the two active sites. The excitation wavelength dependence of the RR data in the low energy shift region (<900 cm⁻¹) for diferric transferrin (Fe₂Tf) reveals extensive changes in the relative intensities for some of the peaks, indicating that the visible and near ultraviolet absorption of the Fe₂Tf protein is composed of several distinct transitions. The identity of the low-energy vibrations was explored by comparison of the data from Fe₂Tf, two different binding site mutants of the N-terminal site half transferrin molecule, Tf/2N, and Fe₂Tf in which the normal binding site carbonate was replaced with C¹⁸O₃²⁻. The higher energy RR spectra of the various samples are quite similar, whereas the low-energy band patterns are strongly influenced by the mutations and isotopic substitution. Comparison of the RR data obtained from Fe₂Tf, Tf/2N, and C-terminal monoferric transferrin reveals that the intensities and energies of the modes below 900 cm⁻¹ are different for the two binding sites. This result helps reveal an isolated electronic transition for the N-terminal active site near 365 nm, where laser excitation yields selective enhancement of the low-energy N-terminal modes. © 1997 John Wiley & Sons, Inc. Biospectroscopy 3: 435–444, 1997