Soybean lipoxygenase-1 oxidizes 3Z-nonenal. A route to 4s-hydroperoxy-2e-nonenal and related products

Abstract

In previous work with soybean (Glycine max), it was reported that the initial product of 3Z-nonenal (NON) oxidation is 4-hydroperoxy-2E-nonenal (4-HPNE). 4-HPNE can be converted to 4-hydroxy-2E-nonenal by a hydroperoxide-dependent peroxygenase. In the present work we have attempted to purify the 4-HPNE-producing oxygenase from soybean seed. Chromatography on various supports had shown that O2 uptake with NON substrate consistently coincided with lipoxygenase (LOX)-1 activity. Compared with oxidation of LOX's preferred substrate, linoleic acid, the activity with NON was about 400- to 1000-fold less. Rather than obtaining the expected 4-HPNE, 4-oxo-2E-nonenal was the principal product of NON oxidation, presumably arising from the enzyme-generated alkoxyl radical of 4-HPNE. In further work a precipitous drop in activity was noted upon dilution of LOX-1 concentration; however, activity could be enhanced by spiking the reaction with 13S-hydroperoxy-9Z, 11E-octadecadienoic acid. Under these conditions the principal product of NON oxidation shifted to the expected 4-HPNE. 4-HPNE was demonstrated to be 83% of the 4S-hydroperoxy-stereoisomer. Therefore, LOX-1 is also a 3Z-alkenal oxygenase, and it exerts the same stereospecificity of oxidation as it does with polyunsaturated fatty acids. Two other LOX isozymes of soybean seed were also found to oxidize NON to 4-HPNE with an excess of 4S-hydroperoxy-stereoisomer.

Figure 1

Coelution of LOX-1 activity (linoleic acid substrate) with NON-oxidizing activity from DEAE-Sephadex. O₂-electrode assay. □, Linoleic acid assay, μmol min⁻¹ mL⁻¹; •, NON, μmol min⁻¹ mL⁻¹.

Figure 2

pH dependence for oxidation of NON by LOX-1 measured by O₂ electrode. Conditions were: 0.2 m buffer, 0.1 mm NON, and 400 μL of a 1.1 m (NH₄)₂SO₄ solution of LOX-1 (2.4 mg of protein) incubated at 25°C in a total volume of 2.4 mL. Buffers were potassium acetate, pH 5.1; potassium Mes, pH 6.1; potassium Pipes, pH 7.0; potassium Hepes, pH 7.9, potassium borate, pH 8.5 and 8.9; and potassium carbonate, pH 9.2 and 10.0.

Figure 3

HPLC separation of products of NON oxidation. LOX-1 added, left to right: none, 0.12 mg, 0.30 mg, and 0.59 mg. NON (1 mm) was incubated for 15 min on ice under pure O₂ in 2 mL of 50 mm potassium borate buffer, pH 8.3. UV detection occurred at 226 nm. Samples of equivalent size were analyzed; values assigned to peaks are relative peak areas. Abbreviations and retention times are: 4-ONE, 9.4 min; 4-HPNE, 24.8 min; and 4-HNE, 53 min.

Figure 4

Nonlinearity of NON-oxidizing activity with amount of LOX-1 added illustrating the threshold requirement (▪), compared with the same conditions plus 55 μg (0.09 mm) of 13S-HPODE (○). NON (1 mm) in 0.1 m potassium borate buffer (pH 8.6) was incubated for 5 min on ice in the presence of pure O₂; reaction volume was 2 mL.

Figure 5

O₂-electrode measurement of nonlinearity of NON-oxidizing activity versus amount of LOX-1 added illustrating the threshold requirement. Initial rate was measured. Conditions: 0.1 m potassium borate buffer, pH 8.6, 1 mm NON, and varying amounts of LOX-1 suspended in 2.3 m (NH₄)₂SO₄ (amount added was kept constant at 90 μL) incubated at 25°C in a total volume of 2.4 mL.

Figure 6

HPLC separation of NON oxidation products after addition of a below-threshold amount of LOX-1 (left), and an identical treatment spiked with 55 μg of 13S-HPODE (right). Conditions, methods, and abbreviations are the same as in Figure 3.

Figure 7

Chiral analysis by GC separation of R- and S-isomers of (-)-menthoxycarbonyl derivatives of methyl 2-hydroxyheptanoates obtained from 4-HNE by chemical modification. Top, Analysis of synthetic 4-HNE; bottom, analysis of 4-HNE derived from LOX-1 oxidation of NON.

Figure 8

Mechanism of LOX oxidation of linoleic acid (top) compared with oxidation of NON (bottom) showing the redox cycling of the Fe active site and the hydrophobic pocket (hatched marks) that accommodates the ω6 tail-end of linoleic acid or NON.