Candadai S. Ramadoss

Double dioxygenation of arachidonic acid by soybean lipoxygenase-1

Abstract Soybean lipoxygenase-1, has been found to catalyze the incorporation of two oxygen molecules into arachidonic acid. The product appears to be 8,15 dihydroperoxy-5,9,11,13-eicosatetraenoic acid. This is apparently the first report of the enzymatic production of a conjugated aliphatic triene in vitro .

Multiple dioxygenation by lipoxygenase of lipids containing all-cis-1, 4, 7-octatriene moieties.

Abstract Soybean lipoxygenase-1 acting upon polyunsaturated fatty acids containing a suitably positioned all- cis -1,4,7-octatriene moiety generates bishydroperoxy derivatives while consuming approximately 2 mol of O 2 per mole of substrate. The reaction has been separated into two steps: The first is marked by the rapid monohydroperoxidation of the starting material and the second is marked by the much slower hydroperoxidation of the first product. All of the compounds which are successfully converted to bishydroperoxy derivatives contain an ω6,9,12 grouping of cis double bonds. α-Linolenic acid (ω3,6,9) cannot serve as a substrate because its preferred site of oxygenation is in the center of the octatriene moiety. The K m for arachidonic acid in the reaction leading to the singly hydroperoxidized monohydroperoxide is 8.6× 10 −5 , m , which is approximately 200 times smaller than the K m for the monohydroperoxide in the second step leading to the bishydroperoxide. All of the substrates which undergo double dioxygenation form conjugated trienes.

Effect of substrate polarity on the activity of soybean lipoxygenase isoenzymes

In order to characterize the several isoenzymes of soybeans, they were examined with respect to the effect of the polar nature of the substrate. In general, lipoxygenase-1 was most active when presented with charged substrates such as the anionic form of linoleic acid or of potassium linoleyl sulfate, whereas lipoxygenase-2 and-3 preferred nonpolar substrates such as unionized linoleic acid, methyl linoleate, linoleyl methane sulfonate, 10,13-nonadecadieneamine, or linoleyl acetate. Linoleyl sulfate, which has been advanced as an excellent readily soluble substrate for lipoxygenase, was indeed the best substrate found for lipoxygenase-1. Lipoxygenase-2 and-3 were, by contrast, totally inactive against this substrate. The favorable response of linnoleic acid to lipoxygenase-2 and-3 at pH 6.8 was ascribed to the anomalously high pKa value of linoleic acid compared to that of short chain carboxylic acids. The pH-activity profile obtained with lipoxygenase acting on linoleyl sulfate (which was charged at all pH values examined) was shifted to lower pH values compared to the linoleic acid activity profile. The effect of changing from the charged to the uncharged substrate, when tested against lipoxygenase-1, was to increase the Km by an order of magnitude.

Coupled oxidation of carotene by lipoxygenase requires two isoenzymes

Abstract The coupled bleaching of carotene observed during the lipoxygenase-catalyzed hydroperoxidation of polyunsaturated fats had previously been shown in our laboratory to be very poorly catalyzed by pure lipoxygenase-1 of soybean. It has now been shown that the other two major isoenzymes in soybean, lipoxygenase-2 and -3, are also individually poor catalysts for the bleaching. However, a combination of lipoxygenase-1 plus lipoxygenase-3, or lipoxygenase-2 plus lipoxygenase-3 are effective in promoting the concomitant oxidation of carotene. Preformed 13-hydroperoxy-9- cis -11- trans octadecadienoate obtained by the action of lipoxygenase-1 on linoleic acid or its ester can replace lipoxygenase-1, when lipoxygenase-3 is present. However, the 9-hydroperoxy-10- trans -12- cis -isomer produced enzymatically with potato lipoxygenase is relatively ineffective.

Effect of albumin on binding and recovery of enzymes in affinity chromatography on Cibacron Blue

Bovine serum albumin appears to improve the specificity of Cibacron Blue F3GA in affinity chromatography of enzymes which interact with nucleotides. The action of bovine serum albumin may rest in its ability to selectively mask affinity sites in the dye, which are not specific for the nucleotide-binding region of the enzyme, while not seriously impairing binding nor its elution by nucleotides. Thus, the elution of Chlorella nitrate reductase from a Blue Sepharose chromatographic column by its coenzyme, NADH, fails, unless the column is first treated with bovine serum albumin. Such treatment also improves the recovery of some other nucleotide-binding enzymes tested.

High-performance liquid chromatographic separation of lipoxygenase isozymes in crude soybean extracts

Abstract A mixture of the soybean lipoxygenase isozymes purified by conventional methods was readily resolved by high-performance liquid chromatography using a SynChropak AX-300 anion-exchange column. Analysis of crude soybean extract by this procedure showed the presence of four different lipoxygenase activities. Mutant soybeans lacking in the isozymes lipoxygenase-1 and -3 were used to test the application of this procedure.

Synthesis of 9(12)-oxy-8,11,15-trihydroxyeicosa-5,13-dienoic acid from arachidonic acid by soybean lipoxygenase-2

Abstract Lipoxygenase-2, an isoenzyme of soybean lipoxygenase catalyzes the formation of a polyhydroxylated furan, 9(12)-oxy-8,11,15-trihydroxyeicosa-5,13-dienoic acid from arachidonic acid.