title: Prostaglandin H synthase kinetics in the two-phase aqueous-micellar system.
abstract: Reaction mixture for PGHS (prostaglandin-H-synthase) is a two-phase system including micellar hydrophobic phase and hydrophilic aqueous phase. Reagents added to the mixture are distributed between phases, thus concentrations of reagents dissolved in phases can differ significantly from their overall contents. Using dynamic light scattering we found that the hydrophobic phase produced by tween-20 consists of micelles, which radius (4-5nm) does not depend on either tween-20 overall content (0.1%-1% v/v) or arachidonic acid (AA) addition (10-1000μM) or PGHS addition (1μM). Tween-20 overall content changing from 0.1% to 2% v/v dramatically affected COX kinetic, but accounting AA distribution between phases allowed us to estimate "true" parameters, independent of the tween-20 overall content and the concentration of another substrate: KM(Ox) equals 9.8μM O2 in the aqueous phase or 0.0074bar in the gaseous phase, KM(AA) equals 5400μM AA in the phase of tween-20 micelles and 5400/PμM AA in the aqueous phase (P is the distribution ratio for the AA between the aqueous phase and the hydrophobic phase (P≫1000)). This approach allowed to evaluate PS, the distribution ratio for the AA between the hydrophobic phase and the PGHS active center (PS ~310). This coefficient indicates the AA selectivity toward the cyclooxygenase active center.
authors.affiliation: International Biotechnological Center of Lomonosov Moscow State University, Russia. Electronic address: ponomareva489@gmail.com.
authors.forename: Olga A
authors.initials: OA
authors.lastname: Ponomareva
authors.affiliation: International Biotechnological Center of Lomonosov Moscow State University, Russia. Electronic address: hydrodiction@gmail.com.
authors.forename: Nikita A
authors.initials: NA
authors.lastname: Trushkin
authors.affiliation: International Biotechnological Center of Lomonosov Moscow State University, Russia. Electronic address: i.s.filimonov.msu@gmail.com.
authors.forename: Ivan S
authors.initials: IS
authors.lastname: Filimonov
authors.affiliation: International Biotechnological Center of Lomonosov Moscow State University, Russia. Electronic address: krivoshey.alexr@gmail.com.
authors.forename: Alexandr V
authors.initials: AV
authors.lastname: Krivoshey
authors.affiliation: International Biotechnological Center of Lomonosov Moscow State University, Russia. Electronic address: barkhatovv@yandex.ru.
authors.forename: Vladimir I
authors.initials: VI
authors.lastname: Barkhatov
authors.affiliation: International Biotechnological Center of Lomonosov Moscow State University, Russia. Electronic address: mitroser04@mail.ru.
authors.forename: Sergey I
authors.initials: SI
authors.lastname: Mitrofanov
authors.affiliation: International Biotechnological Center of Lomonosov Moscow State University, Russia. Electronic address: biocentr@list.ru.
authors.forename: Petr V
authors.initials: PV
authors.lastname: Vrzheshch
mesh_terms: D000818:Animals; D016718:Arachidonic Acid / Q000737:chemistry*; D057927:Hydrophobic and Hydrophilic Interactions; D008027:Light; D008297:Male; D008823:Micelles; D011136:Polysorbates / Q000737:chemistry*; D011451:Prostaglandin-Endoperoxide Synthases / Q000737:chemistry*; D051381:Rats; D012542:Scattering, Radiation
chemical_list: D008823:Micelles; D011136:Polysorbates; D016718:Arachidonic Acid; D011451:Prostaglandin-Endoperoxide Synthases
keywords: Arachidonic acid distribution coefficient; Cyclooxygenase kinetics; Prostaglandin H synthase; Tween-20 micelle size; Two-phase system; “true” Michaelis constants