A simple and efficient method for hemoglobin removal from mammalian tissue cytosol by zinc sulfate and its application to the study of lipoxygenase.
A simple and efficient method is described to remove hemoglobin (Hb) from human term placental cytosol to study dioxygenase and co-oxidase activities of lipoxygenase. In the untreated samples, 70%-80% of the linoleic acid-dependent dioxygenase and co-oxidase activities were found to be associated with the pseudo-lipoxygenase activity of Hb. Zinc sulfate (0.5 mM) precipitated >97% of the Hb present in the cytosol. The dioxygenase activity of the ZnSO4 treated cytosol exhibited a Vmax value of 313 nmoles linoleic acid hydroperoxide formed/min/mg protein and a K(M) of 1.4 mM for linoleic acid. The ZnSO4 treated cytosol displayed co-oxidase activity toward benzidine, dimethoxybenzidine, guaiacol, pyrogallol, tetramethylbenzidine and tetramethyl-p-phenylenediamine. Nordihydroguaiaretic acid, 5,8,11-eicosatriynoic acid, butylated hydroxyanisole, butylated hydroxytoluene and gossypol caused concentration dependent inhibition of dioxygenase and co-oxidase activities. These results suggest ZnSO4 precipitation of Hb from cytosol does not alter the functional characteristics of the human term placental lipoxygenase.
Hover
C G
CG
Florida Toxicology Research Center, Department of Environmental and Occupational Health, College of Public Health, University of South Florida, Tampa 33612-3805, USA.
Kulkarni
A P
AP
D011232:Chemical Precipitation; D002846:Chromatography, Affinity; D003600:Cytosol / Q000737:chemistry* / Q000201:enzymology; D005260:Female; D006454:Hemoglobins / Q000737:chemistry*; D006801:Humans; D019787:Linoleic Acid / Q000378:metabolism; D008084:Lipoxygenase / Q000302:isolation & purification* / Q000378:metabolism; D016859:Lipoxygenase Inhibitors / Q000494:pharmacology; D010920:Placenta / Q000201:enzymology* / Q000648:ultrastructure; D011247:Pregnancy; D019287:Zinc Sulfate
D006454:Hemoglobins; D016859:Lipoxygenase Inhibitors; D019287:Zinc Sulfate; D019787:Linoleic Acid; D008084:Lipoxygenase