DISTINCTION OF THE 2 BINDING-SITES OF SERUM TRANSFERRIN BY RESONANCE RAMAN-SPECTROSCOPY

The resonance Raman (RR) data for a variety of transferrin samples wer e investigated to explore differences between the two active sites. Th e excitation wavelength dependence of the RR data in the low energy sh ift region (<900 cm(-1)) for diferric transferrin (Fe2Tf) reveals exte nsive changes in the relative intensities for some of the peaks, indic ating that the visible and near ultraviolet absorption of the Fe2Tf pr otein is composed of several distinct transitions. The identity of the low-energy vibrations was explored by comparison of the data from Fe2 Tf, two different binding site mutants of the N-terminal site half tra nsferrin molecule, Tf/2N, and Fe2Tf in which the normal binding site c arbonate was replaced with (CO32-)-O-18. 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 substit ution. Comparison of the RR data obtained from Fe2Tf, Tf/2N, and C-ter minal 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 selec tive enhancement of the low-energy N-terminal modes. (C) 1997 John Wil ey & Sons, Inc.