title: Effects of heme-PrP complex on cell-free conversion and peroxidase-linked immunodetection of prions in blood-based assays.
abstract: Prion protein (PrP) binding to natural and synthetic porphyrins has been previously demonstrated but the effects of endogenous heme interactions with PrP remain uncertain. This study investigated implications of this interaction in blood-based peroxidase-linked prion immunodetection and seeded conversion of cellular prion (PrP(C)) into disease associated form (PrP(Sc)). Heme binding to recombinant PrP(C) enhanced intrinsic peroxidase activity (POD) by 2.5-fold and POD inherent to denatured blood accounted for over 84% of luminol-based substrate oxidation in a prion immunodetection assay. An immuno-capture assay showed that 75-98% of blood POD was attributable to binding of PrP(C) with endogenous heme. Additionally, 10 μM heme inhibited (P<0.05) the seeded conversion of PrP(C) to PrP(Sc) through the protein misfolding cycling amplification assay. We conclude that the observed effects can interfere with cell-free conversion and peroxidase-linked immunodetection of prions in blood-based assays. These results indicate that heme-PrP interactions could modulate intrinsic POD and protect PrP(C) from conversion into PrP(Sc).
authors.affiliation: Ottawa Laboratory (Fallowfield), Canadian Food Inspection Agency, Ottawa, Ontario, Canada. Electronic address: andrei.soutyrine@inspection.gc.ca.
authors.forename: Andrei
authors.initials: A
authors.lastname: Soutyrine
authors.affiliation: Ottawa Laboratory (Fallowfield), Canadian Food Inspection Agency, Ottawa, Ontario, Canada.
authors.forename: Nishandan
authors.initials: N
authors.lastname: Yogasingam
authors.affiliation: Ottawa Laboratory (Fallowfield), Canadian Food Inspection Agency, Ottawa, Ontario, Canada.
authors.forename: Hongsheng
authors.initials: H
authors.lastname: Huang
authors.affiliation: Ottawa Laboratory (Fallowfield), Canadian Food Inspection Agency, Ottawa, Ontario, Canada.
authors.forename: Gordon
authors.initials: G
authors.lastname: Mitchell
mesh_terms: D000818:Animals; D006418:Heme / Q000378:metabolism*; D008165:Luminol; D046912:Multiprotein Complexes / Q000378:metabolism*; D010084:Oxidation-Reduction; D009195:Peroxidase / Q000378:metabolism*; D018621:PrPC Proteins / Q000737:chemistry / Q000378:metabolism*; D018620:PrPSc Proteins / Q000737:chemistry / Q000302:isolation & purification*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D046912:Multiprotein Complexes; D018621:PrPC Proteins; D018620:PrPSc Proteins; D011994:Recombinant Proteins; D006418:Heme; D008165:Luminol; D009195:Peroxidase
keywords: Blood-based prion detection assays; Heme–PrP complex; Peroxidase; Prion protein conversion