title: Tris(3-hydroxypropyl)phosphine is superior to dithiothreitol for in vitro assessment of vitamin K 2,3-epoxide reductase activity.
abstract: Use of the reductant dithiothreitol (DTT) as a substrate for measuring vitamin K 2,3-epoxide reductase (VKOR) activity in vitro has been reported to be problematic because it enables side reactions involving the vitamin K1 2,3-epoxide (K1>O) substrate. Here we characterize specific problems when using DTT and show that tris(3-hydroxypropyl)phosphine (THPP) is a reliable alternative to DTT for in vitro assessment of VKOR enzymatic activity. In addition, the pH buffering compound imidazole was found to be problematic in enhancing DTT-dependent non-enzymatic side reactions. Using THPP and phosphate-based pH buffering, we measured apparent Michaelis-Menten constants of 1.20 μM for K1>O and 260 μM for the active neutral form of THPP. The Km value for K1>O is in agreement with the value that we previously obtained using DTT (1.24 μM). Using THPP, we successfully eliminated non-enzymatic production of 3-hydroxyvitamin K1 and its previously reported base-catalyzed conversion to K1, both of which were shown to occur when DTT and imidazole are used as the reductant and pH buffer, respectively, in the in vitro VKOR assay. Accordingly, substitution of THPP for DTT in the in vitro VKOR assay will ensure more accurate enzymatic measurements and assessment of warfarin and other 4-hydroxycoumarin inhibition constants.
authors.affiliation: Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, 60348 Frankfurt-am-Main, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Krettler
authors.affiliation: Im Hermeshain 6, 60388 Frankfurt-am-Main, Germany.
authors.forename: Carville G
authors.initials: CG
authors.lastname: Bevans
authors.affiliation: Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, 60348 Frankfurt-am-Main, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Reinhart
authors.affiliation: Institute of Experimental Haematology and Transfusion Medicine, University Clinic Bonn, 53105 Bonn, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Watzka
authors.affiliation: Institute of Experimental Haematology and Transfusion Medicine, University Clinic Bonn, 53105 Bonn, Germany. Electronic address: johannes.oldenburg@ukb.uni-bonn.de.
authors.forename: Johannes
authors.initials: J
authors.lastname: Oldenburg
mesh_terms: D055162:Biocatalysis; D002021:Buffers; D002793:Cholic Acids / Q000378:metabolism; D004229:Dithiothreitol / Q000378:metabolism*; D057075:Enzyme Assays; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010084:Oxidation-Reduction; D010720:Phosphines / Q000378:metabolism*; D010843:Pichia / Q000378:metabolism; D019163:Reducing Agents / Q000378:metabolism; D012996:Solutions; D013379:Substrate Specificity; D064417:Vitamin K Epoxide Reductases / Q000378:metabolism*
chemical_list: D002021:Buffers; D002793:Cholic Acids; D004229:Dithiothreitol; D010720:Phosphines; D019163:Reducing Agents; D012996:Solutions; D064417:Vitamin K Epoxide Reductases; C000603174:tris(3-hydroxypropyl)phosphine; C080938:tris(2-carboxyethyl)phosphine; C044646:phosphine; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate
keywords: Dithiothreitol (DTT); In vitro assay; Tris(3-hydroxypropyl)phosphine (THPP); Vitamin K 2,3-epoxide reductase (VKOR) activity; Vitamin K 2,3-epoxide reductase complex subunit 1 (VKORC1); Warfarin