207 - Redox Activity Induced by Metal Coordination with Prion Peptide

Abstract

Background Physiological function of prion protein (PrP) is still unknown, however its redox and metal-reservoir potentials are viewed as meaningful. We also showed the conformational change of the human PrP binding to Cu2+ [1]. In this report, we have examined the redox ability and the stability of metals at binding site using octarepeat peptide, which is a primary site for Cu2+ uptake in the N-terminal region of PrP. Materials and Methods An eight-residue peptide (PHGGGWGQ) of prion protein was used for the spectroscopic affinity assay for metal ions (Cu2+, Ni2+, Co2+, Zn2+, and Mn2+). For electron spin resonance (ESR), the peptide and Cu2+ were prepared as 0.1 mM in 10 mM HEPES buffer (pH 7.6) containing 30% glycerol. The ESR spectrum was acquired at 20 K. For circular dichroism (CD), the peptide and Cu2+ were prepared as 0.8 mM in 15 mM HEPES buffer. To verify competitive metal binding, octapeptide-Cu2+ was titrated with other divalent metal ions such as Ni2+, Co2+, Zn2+, and Mn2+. The reactivity of metal-containing peptide with ROS was analyzed by ESR spin trapping with CYPMPO. Results The ESR at 20K showed two types of coordination modes for the binding of octapeptide-Cu2+. The CD spectrum of octapepide-Cu2+ indicated the characteristic positive peak of molecular ellipticity at visible wavelengths, which was also assigned to Cu2+ coordination complexes. The CD peak of octapeptide-Cu2+was reduced by titrating with other divalent metal ions, such as Ni2+, Co2+, Zn2+, and Mn2+. ESR spin trapping showed that the copper-peptide complex converted superoxide into hydroxyl radical.