Hemin and hemeprotein bleaching during linoleic acid oxidation by lipoxygenases.
Hemin and hemoglobin are bleached by lipoxygenases, type 1 (from soybean) or type 2 (from platelets), during linoleic acid oxidation. This process has been found to be related to the inhibition of the lipoxygenase activity, measured as hydroperoxide generation and to produce oxodienes as well. All these parameters have been determined simultaneously from measurements of the absorbance at 234, 285, 375 and 410 nm to detect hydroperoxides, oxodienes, hemin and hemoglobin, respectively, using a diode array spectrophotometer. The inhibition of lipoxygenase activity by these pigments has been found to be competitive with linoleic acid, showing an increase of 4-7-fold of the Km value of linoleic acid in the presence of concentrations of hemin and hemoglobin as low as 0.2 and 0.02 microM, respectively, for the case of platelet lipoxygenase activity. The concentrations of hemin and of hemoglobin producing the inhibition of 50% of lipoxygenase activity are: 0.25 and 0.02 microM for the platelet isoenzyme, and 1.4 and 0.18 microM for the soybean isoenzyme, respectively. From the quenching of the intrinsic fluorescence of soybean lipoxygenase activity by hemin, we have obtained a dissociation constant of hemin-soybean lipoxygenase of 0.5 microM. The results obtained in this paper for the cooxidation process of hemin and hemoglobin by lipoxygenase can be rationalized in terms of hemin binding at or near to the catalytic center, resulting in a lesser binding of linoleic acid and an enhanced release of radicals, and pigment bleaching by radicals and lipid hydroperoxides.
Macías
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Dpto. de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de Extremadura, Badajoz, Spain.
Pinto
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Gutiérrez-Merino
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D000818:Animals; D001792:Blood Platelets / Q000201:enzymology; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D005454:Fluorescence Polarization; D006420:Hemeproteins / Q000737:chemistry*; D006427:Hemin / Q000737:chemistry*; D006454:Hemoglobins / Q000737:chemistry*; D006861:Hydrogen Peroxide / Q000737:chemistry; D007527:Isoenzymes / Q000737:chemistry*; D007700:Kinetics; D019787:Linoleic Acid; D008041:Linoleic Acids / Q000737:chemistry*; D008084:Lipoxygenase / Q000737:chemistry*; D010084:Oxidation-Reduction; D010860:Pigments, Biological; D051381:Rats; D011919:Rats, Inbred Strains; D013025:Glycine max / Q000201:enzymology
D006420:Hemeproteins; D006454:Hemoglobins; D007527:Isoenzymes; D008041:Linoleic Acids; D010860:Pigments, Biological; D006427:Hemin; D019787:Linoleic Acid; D006861:Hydrogen Peroxide; D008084:Lipoxygenase