Prostaglandin H synthase kinetics in the two-phase aqueous-micellar system

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...

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Bibliographic Details
Published in:	Biochimica et biophysica acta Vol. 1858; no. 9; pp. 2199 - 2207
Main Authors:	Ponomareva, Olga A., Trushkin, Nikita A., Filimonov, Ivan S., Krivoshey, Alexandr V., Barkhatov, Vladimir I., Mitrofanov, Sergey I., Vrzheshch, Petr V.
Format:	Journal Article
Language:	English
Published:	Netherlands Elsevier B.V 01-09-2016
Subjects:	Animals Arachidonic Acid - chemistry Arachidonic acid distribution coefficient Cyclooxygenase kinetics Hydrophobic and Hydrophilic Interactions Light Male Micelles Polysorbates - chemistry Prostaglandin H synthase Prostaglandin-Endoperoxide Synthases - chemistry Rats Scattering, Radiation Tween-20 micelle size Two-phase system “true” Michaelis constants AA KM PGHS DEAE Sepharose TMPD “true” Michaelis constants DEDTC Two-phase system COX reaction Tween-20 micelle size Arachidonic acid distribution coefficient Vm IC50 Cyclooxygenase kinetics disodium EDTA POX reaction Prostaglandin H synthase
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Summary:	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: KMOx equals 9.8μM O2 in the aqueous phase or 0.0074bar in the gaseous phase, KMAA 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. [Display omitted] •Tween-20 micelle radius in the reaction mixture equals 4–5nm.•The distribution ratio of AA between the aqueous phase and the micelles P≫1000•“True” KMOx equals 9.8μM O2 in aqueous phase or 0.0074bar in the gaseous phase.•“True” KMAA equals 5400μM AA in the micelles or 5400/PμM AA in the aqueous phase.
ISSN:	0005-2736 0006-3002 1879-2642
DOI:	10.1016/j.bbamem.2016.06.013