A novel crosslinking reagent and its application for the detection and isolation of heparin-binding protein(s) on the platelet surface.
A new hetero-bifunctional photo crosslinking reagent, 2-(4-azidoanilyl)-4-(4-azabicyclo-[2,2, 2]hexylammonio)-6-morpholino-1,3,5-triazine chloride, was designed to detect and isolate heparin-binding protein(s) that may act as heparin-receptor(s) on the platelet surface. In a preliminary study using ethanol as a model substrate, the reagent was shown to react with the alcoholic hydroxy group under mild conditions and its crosslinking photoreactivity was high. The reagent effectively formed similar covalent bonds with heparin, while preserving its anticoagulant anti-Xa activity. [3H]Heparin labeled with this reagent crosslinked to antithrombin III very specifically but not to ovalbumin, as analyzed by the Bio-imaging Analyzer System (BAS, Fuji Photo Film, Tokyo). Affinity crosslinking of [3H]heparin was then used to detect heparin-binding proteins on the surface of intact platelets. Several discrete protein bands were detected by the BAS-imaging of SDS-PAGE.
Suda
Y
Y
Department of Chemistry, Graduate School of Science, Osaka University, Toyonaka, Osaka, 560-0043, Japan. ysuda@chem.sci.osaka-u.ac.jp
Mori
K
K
Bird
K
K
Marques
D
D
Ormsby
J
J
Tanaka
S
S
Koshida
S
S
Nakamura
M
M
Kusumoto
S
S
Sobel
M
M
D000438:Alcohols; D000990:Antithrombin III; D001792:Blood Platelets / Q000737:chemistry* / Q000378:metabolism; D001798:Blood Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002352:Carrier Proteins / Q000097:blood / Q000302:isolation & purification*; D003432:Cross-Linking Reagents / Q000138:chemical synthesis; D006493:Heparin / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008565:Membrane Proteins / Q000097:blood / Q000302:isolation & purification*; D009025:Morpholines / Q000138:chemical synthesis; D010047:Ovalbumin
C122046:2-(4-azidoanilyl)-4-(4-azabicyclo-(2,2,2)hexylammonio)-6-morpholino-1,3,5-triazine chloride; D000438:Alcohols; D001798:Blood Proteins; D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D008565:Membrane Proteins; D009025:Morpholines; D000990:Antithrombin III; D006493:Heparin; D010047:Ovalbumin