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

Kazuki Takeda; Ayuko Morita; Yoshinori Ikenaka; Shouta M M Nakayama; Mayumi Ishizuka

doi:10.1016/j.vas.2020.100095

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

Vet Anim Sci. 2020 Feb 15:9:100095. doi: 10.1016/j.vas.2020.100095. eCollection 2020 Jun.

Authors

Kazuki Takeda¹, Ayuko Morita¹, Yoshinori Ikenaka^{1

2}, Shouta M M Nakayama¹, Mayumi Ishizuka¹

Affiliations

¹ Department of Environmental Sciences, Laboratory of Toxicology, Graduate School of Veterinary Medicine, Hokkaido University, Kita-18 Nishi-9, Kita-ku, Sapporo 060-0818, Japan.
² Water Research Group, Unit for Environmental Sciences and Management, North-West University, Potchefstroom, South Africa.

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 IC₅₀ for warfarin.

Keywords: DTT, Dithiothreitol; Dithiothreitol; ER, endoplasmic reticulum; THP, tris(3-hydroxypropyl)phosphine; Tris(3-hydroxypropyl)phosphine; VK, vitamin K quinone; VKO, vitamin K epoxide; VKOR, Vitamin K epoxide reductase; Vitamin K; Vitamin K epoxide reductase; anticoagulant.