Enzymic oxidation of carotene and linoleate by alfalfa: Properties of active fractions

Abstract

An aqueous Triton X-100 extract, prepared from alfalfa leaves, was fractionated on carboxymethylcellulose into four fractions designated A–D. Fractions B and C, which contained the bulk of the carotene-bleaching and linoleate-oxidizing activity, resembled soyben lipoxygenase in several respects. The most striking among these were the high specific activities with regard to the formation of conjugated dienes and oxygen absorption by linoleate; the inability to oxidize oleate; and the insensitivity to cyanide. Fraction C, like lipoxygenase, exhibited the absorption spectrum of a pure protein devoid of a prosthetic chromophore, whereas the spectrum of fraction B hinted at some contamination with a heme protein. Fraction A resembled peroxidase in its high specific peroxidase and indoleacetic acid oxidase activities, coupled with weak linoleate oxidizing and carotene-bleaching activities. Like peroxidase, fraction A was able to effect some bleaching of carotene in the presence of oleate and also in the absence of polyunsaturated fatty acids, with a characteristic rapid decline in activity. Additional points of resemblance of fraction A with peroxidase were its sensitivity to cyanide and ascorbic acid and its pH optimum in the carotene-bleaching test. Finally, fraction A could be separated on carboxymethylcellulose into two subfractions, which exhibited heme protein absorption spectra with typical redox shifts. Fraction D was only weakly active. It exhibited some of the properties of heme proteins, but no clear picture as to its nature could be obtained by the tests used.