Anomalous oxidation of MDL 73,404 by horseradish peroxidase.
3,4-Dihydro-6-hydroxy-N,N,N-2,5,7,8-heptamethyl-2H-1-benzopyran-2-ethanaminium-4-methylbenzene sulfonate (MDL 73,404) is a cardioselective water-soluble quaternary ammonium analogue of Vitamin E which is synthesized to augment the antioxidant defence in situations of free radical injury such as myocardial infarction/reperfusion. Its oxidation by any peroxidative enzyme has not been studied kinetically. This paper describes its enzymatic oxidation by horseradish peroxidase (HRP). The activity was followed spectrophotometrically at 255nm, and the experimental results were simulated using the program "KINETIC 3.1" for Windows 3.x. The MDL 73,404 was oxidized by horseradish peroxidase in the presence of H2O2 to its corresponding MDL 73,404 quinone. During this oxidation, the horseradish peroxidase showed an unexpectedly slow kinetic response with time, which contrast with the linear product accumulation curve measured with 2,2'-azino-bis-(3-estilbenzotiazol-6-sulfonic acid) (ABTS). This response was dependent on the respective concentrations of enzyme, MDL 73,404 and H2O2. However, when the enzyme was incubated with H2O2, the slow kinetic response disappeared and a lag period was observed. Furthermore, when p-coumaric acid (PCA) was added, the activity increased and the slow kinetic response became a straight line. In order to explain this anomalous behaviour, a kinetic model has been proposed and its differential equations simulated. From the correlation between experimental and simulated results it is concluded that MDL 73,404 can act as a slow response substrate for peroxidase, probably due to the presence of a quaternary ammonium side chain that confers on it a slow capacity to convert compound III into ferriperoxidase.
Núñez-Delicado
Estrella
E
Department of Biochemistry and Molecular Biology-A, Faculty of Biology, University of Murcia, Campus de Espinardo, E-30071, Murcia, Spain
Sojo
Mar
M
García-Carmona
Francisco
F
Sánchez-Ferrer
Alvaro
A
D003373:Coumaric Acids / Q000737:chemistry / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000737:chemistry* / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000737:chemistry; D007700:Kinetics; D016014:Linear Models; D010084:Oxidation-Reduction; D011422:Propionates; D011809:Quinones / Q000737:chemistry; D013053:Spectrophotometry / Q000379:methods; D014810:Vitamin E / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism
D003373:Coumaric Acids; D011422:Propionates; D011809:Quinones; C067190:MDL 73404; D014810:Vitamin E; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase; C495469:p-coumaric acid