Metal-binding ability of human prion protein fragment peptides analyzed by column switch HPLC

Abstract

The structural conversion of the prion protein (PrP) from the normal cellular isoform (PrP C) to the posttranslationally modified form (PrP Sc) is thought to relate to Cu 2+ binding to histidine (H) residues. Traditionally, the binding of metals to PrP has been investigated by monitoring the conformational conversion using circular dichroism (CD). In this study, the metal-binding ability of 21 synthetic peptides representing regions of human PrP C was investigated by column switch high-performance liquid chromatography (CS-HPLC). The CS-HPLC system is composed of a metal chelate affinity column and an octadecylsilica (ODS) reversed-phase column that together enable the identification of metal-binding regardless of conformational conversion. Synthetic peptides were designed with respect to the position of H residues as well as the secondary structure of human PrP (hPrP). The ability of the octapeptide (PHGGGWGQ)-repeating region (OP-repeat) to bind metals was analyzed by CS-HPLC and supported by CD analysis, and indicated that CS-HPLC is a reliable and useful method for measuring peptide metal-binding. Peptides from the middle region of hPrP showed a high affinity for Cu 2+, but binding to Zn 2+, Ni 2+, and Co 2+ was dependent on peptide length. C-Terminal peptides had a lower affinity for Cu 2+, Zn 2+, Ni 2+, and Co 2+ than OP-repeat region peptides. Interestingly, hPrP193 - 230, which contained no H residues, also bound to Cu 2+, Zn 2+, Ni 2+, and Co 2+, indicating that this region is a novel metal-binding site in the C-terminal region of PrP C. The CS-HPLC method described in this study is useful and convenient for assessing metal-binding affinity and characterizing metal-binding peptides or proteins. © 2011 Pharmaceutical Society of Japan.