title: Development of a photoreactive probe-based system for detecting heparin.
abstract: We previously identified a peptide heparin-associated peptide Y (HappY) that binds specifically to heparin. In this article, we report a novel heparin detection system using chemically modified HappY as a probe. The photoreactive HappY probe was serially diluted and dispensed into a 96-well plate coated with biotinylated heparin. After ultraviolet irradiation, the HappY probe crosslinked to the heparin on the plate was detected with fluorescein isothiocyanate-conjugated streptavidin. Furthermore, the photoreactive HappY probe was used to stain cutaneous tissue sections obtained from dermatitis-affected or mastocytoma-affected cats and dogs. The photoreactive HappY probe stained limited resident mast cells in the connective tissue of skin compared with the anti-heparan sulfate monoclonal antibody 10E4, suggesting that the probe can be used to distinguish the structure of heparin in tissues. The interactions between glycosaminoglycans and proteins in vivo tend to be weak. Therefore, our method for enhancing such weak interactions may be a promising tool for intermolecular interaction studies in glycobiology research.
authors.affiliation: Department of Applied Life Science, Faculty of Applied Biological Sciences, Gifu University, Gifu 501-1193, Japan. Electronic address: yabet@gifu-u.ac.jp.
authors.forename: Tomio
authors.initials: T
authors.lastname: Yabe
authors.affiliation: Department of Applied Life Science, Faculty of Applied Biological Sciences, Gifu University, Gifu 501-1193, Japan; Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.
authors.forename: Ritsuko
authors.initials: R
authors.lastname: Hosoda-Yabe
authors.affiliation: Department of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University, Gifu 501-1193, Japan.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Sakai
authors.affiliation: Department of Applied Life Science, Faculty of Applied Biological Sciences, Gifu University, Gifu 501-1193, Japan.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Kanamaru
authors.affiliation: Department of Applied Life Science, Faculty of Applied Biological Sciences, Gifu University, Gifu 501-1193, Japan; Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Kiso
mesh_terms: D000818:Animals; D002415:Cats; D003872:Dermatitis / Q000378:metabolism* / Q000473:pathology; D004285:Dogs; D019793:Fluorescein / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry*; D006493:Heparin / Q000378:metabolism*; D008407:Mast Cells / Q000378:metabolism* / Q000473:pathology; D054705:Mastocytoma, Skin / Q000378:metabolism* / Q000473:pathology; D010455:Peptides / Q000737:chemistry*
chemical_list: D005456:Fluorescent Dyes; D010455:Peptides; D006493:Heparin; D019793:Fluorescein
keywords: Detection; Heparin; Mastocytoma; Photoreactive probe