The overall objective of this research is to develop a method to isolate and stabilize TXA2, a potent constrictor of vascular smooth muscle and an important mediator of the platelet release reaction, and provide direct spectroscopic information on its structure. Preliminary studies have been performed to address three pertinent issues. First, that nonpolar prostaglandins can be rapidly extracted from aqueous solution in their ionized form by use of an exhaustively silylated C18 solid-state extraction system and that in this form labile groups such as the enol ether in PGI2 are protected for several minutes from chemical conditions which would inactivate the molecule in a matter of seconds if in solution. Secondly, that highly purified TXA2 synthase (nearly homogeneous by electrophoresis) can be immobilized both by its adsorption onto phenyl-Sepharose and by encapsulation into a polysiloxane matrix while still retaining a significant portion of its biological activity. Finally, that unstable prostaglandins such as PGH2 can be analyzed by direct chemical ionization (DEP)MS without the need for chemical derivatization. We plan to prepare an online TXA2 synthetic apparatus, similar to a micro HPLC system using the immobilized synthase in a column which we anticipate will produce TXA2 free of protein in less than 30 seconds. The eluate from this column will be injected directly into a MS by a spray-type interface and also examined by DEP probe MS (EI and CI). The eluate from the immobilized synthase will also be connected to a second column consisting of the C18 solid-state extraction system for the isolation and stabilization of TXA2. The solid-state extraction system containing the PGH2 metabolites HHT and TXA2 (TXB2 does not bind) will be dried, extracted with aprotic organic solvents and examined using high field NMR and IR. Attempts will be made to separate HHT and TXA2 using polar bonded phase HPLC and aprotic organic solvents. If separation is not possible the HHT and TXA2 component mixture spectra will be resolved using subtraction techniques.
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