title: 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 Cu²⁺ 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 Cu²⁺, but binding to Zn²⁺, Ni²⁺, and Co²⁺ was dependent on peptide length. C-Terminal peptides had a lower affinity for Cu²⁺, Zn²⁺, Ni²⁺, and Co²⁺ than OP-repeat region peptides. Interestingly, hPrP193-230, which contained no H residues, also bound to Cu²⁺, Zn²⁺, Ni²⁺, and Co²⁺, 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.
authors.affiliation: Department of Analytical Chemistry, Pharmaceutical Science, Setsunan University, Japan.
authors.forename: Aya
authors.initials: A
authors.lastname: Kojima
authors.forename: Yasunori
authors.initials: Y
authors.lastname: Mabuchi
authors.forename: Motomi
authors.initials: M
authors.lastname: Konishi
authors.forename: Rika
authors.initials: R
authors.lastname: Okihara
authors.forename: Makoto
authors.initials: M
authors.lastname: Nagano
authors.forename: Toshifumi
authors.initials: T
authors.lastname: Akizawa
mesh_terms: D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D008670:Metals / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D018621:PrPC Proteins / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding
chemical_list: D008670:Metals; D010446:Peptide Fragments; D018621:PrPC Proteins