Comparison of two reducing agents dithiothreitol and tris(3-hydroxypropyl)phosphine for in vitro kinetic assay of vitamin K epoxide reductase

Abstract

Vitamin K epoxide reductase (VKOR) is a target enzyme for anticoagulants, such as warfarin, that are used as medicines or rodenticides. Assessing VKOR activity is required to ensure the proper usage of these drugs. Dithiothreitol (DTT) is a typical disulfide reductant that is used as a substrate for in vitro VKOR assays. However, DTT is considered problematic because of its side effects. Tris(3-hydroxypropyl)phosphine (THP) has been found to be a reliable alternative to DTT, as shown by kinetic analyses of the VKOR with them. THP showed significantly lower V max and K m values than those of DTT; however, there was no significant difference in their V max / K m and IC 50 for warfarin.

The VKORC1 ER-luminal loop mutation (Leu76Pro) leads to a significant resistance to warfarin in black rats (Rattus rattus)

“…Purification of vitamin Ks and quantification by mass spectrometry were also performed according to the method of Takeda et al [21]. VK and VKO were extracted from the reaction mixture using the liquid-liquid extraction method, and vitamin K1-d7 was added as an internal standard (final concentration, 100 nM).…”

Simultaneous Determination of Vitamin E and Vitamin K in Food Supplements Using Adsorptive Stripping Square-Wave Voltammetry at Glassy Carbon Electrode

“…Vitamin E (VE), known as the most active lipophilic antioxidant, exists as four tocopherol and tocotrienol isomers [3,4,6,7] whose sources include vegetable oils, nuts and seeds of plants [8,9]. Vitamin K present in green plants (phylloquinone; VK1) and produced by bacteria (menaquinone; VK2) [10][11][12][13][14], is widely used in the diet for its anti-hemorrhagic properties [11,12].…”