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 pK_a 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 K_m by an order of magnitude.     

Polycarboxylates in soda ash detergents

Calcium carbonate deposition on cotton fabric from soda ash-based detergents containing polycarboxylates is measured and related to polymer composition and molecular weight, under varying conditions of temperature, water hardness and detergent composition. Deposition effects on fabric are compared with threshold inhibition effects and calcium binding capacities of the polymers. Polyacrylic acids with molecular weights of 2000–5000 are most effective in preventing calcium carbonate deposition on fabric. A 1.5:1 acrylic acid:maleic acid copolymer is more effective than polyacrylic acid. Detergents with LAS left somewhat higher calcium carbonate deposits than detergents with a nonionic surfactant. Prevention of visible calcium carbonate precipitation in the absence of fabric (“threshold effects”) appears at polymer levels considerably lower than those necessary to prevent calcium carbonate deposition on fabric. Deposition on fabric can be prevented at levels of polymer much less than that necessary to bind most of the calcium ions, as determined by the calcium binding capacity. Presented at the AOCS meeting in New Orleans in May 1987.     

Micelle formation in mixtures of nonionic and cationic detergents

The effect of a homologous series of polyoxyethylene n-dodecanols on the critical micelle concentration (cmc) of mixtures with n-dodecyl trimethyl ammonium bromide (DTAB) and of polyoxyethylene n-hexadecanols on the cmc of mixtures with n-hexadecyl trimethyl ammonium bromide (CTAB) has been studied in terms of the composition of the mixtures. The cmc of the nonionic component of the mixed micelles is approximately one hundredth of that of the cationic component. Only a gradual increase in the cmc values of the mixed micelles above the values of the nonionic components was observed in the composition range of 0–75 mole % or 0–90 mole %, respectively, of cationic detergent. This is followed by an abrupt transition to the high cmc values of the cationic component. This abrupt transition is considerably reduced by addition of NaBr. It is postulated that in the absence of added electrolyte the degree of ionic repulsion of the cationic component in mixed micelles is markedly decreased as the proportion of nonionic component reaches a threshold range of 25 or 10 mole %, respectively. Swamping of charges reduces ionic repulsion in NaBr solutions and, consequently, the abrupt transition in the cmc vs. composition curves is reduced.     

Micelle formation in mixtures of nonionic and anionic detergents

This paper describes the effect of a homologous series of polyoxyethylene n-dodecanols on the critical micelle concentration (CMC) of sodium n-dodecyl alcohol sulfate as a function of composition of the mixtures and temperature. The CMC of the nonionic component of the mixed micelles is about one-hundredth of that of the anionic. Only a gradual increase in the CMC values of the mixed micelles above the values of the nonionic components was observed in the composition range of 0–90 mole % anionic detergent. This is followed by an abrupt transition to the high CMC values of the anionic component. The gradual increase of the CMC values in the range below 90 mole % anionic detergent of n-dodecanol+ 4 EO exceeds that of the higher homologs containing 7, 23 and 30 ethylene oxide units. It is postulated that the degree of ionic repulsion of the ionic component in mixed micelles is markedly decreased as the proportion of nonionic component reaches a threshold range of 10 mole %. This effect is more pronounced with large ethylene oxide coils operating at the periphery of the micelle core than with short ethylene oxide coils. Thermodynamic data have been included.     

Physical state of inhibitor fatty acids and linoleate solutions under lipoxygenase assay conditions

A variety of fatty acids, which are potential competitive inhibitors of soybean lipoxygenase (EC 1.13.11.12), give kinetically unstable mixtures in standard assay solutions containing linoleate (substrate). In the assay solution (pH 10, 0.1 M borate, 1.63% ethanol), each fatty acid by itself shows normal surface tension vs concentration behavior; but, despite a range of solubilization techniques in the presence of 10 μM or higher linoleate and low concentrations of these materials, irreproducible surface-tension readings and inhibition kinetics results. This inhomogeneity (or kinetic instability) disappears as the concentration increases. Critical micelle concentration (CMC) values of mixtures are not additive, and binary mixture behavior depends on fatty acid structure. Several lines of observation, including CMC values and actual surface tension (γ) values for several systems, suggest premicellar heterodimer or higher mixed aggregate formation. Lipids with K_i significantly above the irreproducible surface-tension range give good kinetic behavior, and K_i is reported. The results are in accord with earlier work on aspects of these systems. Complementary solution physical studies must be done for any kinetic (or specificity) determinations of enzymes using lipids. The notation for fatty acids follows the system in which a,b-x:y indicates a fatty acid of x carbons with ycis double bonds at positions a and b from the CO₂H;trans olefin is designated by (t) following the position number; SC₁₀S and SC₁₂S are sodium decyl and sodium dodecyl sulfates; 9,12(OH)-18∶1 is ricinoleic acid; CMC is critical micelle concentration. Surface tension is γ.     

Liquid detergents from cationic,anionic and nonionic surfactants. adsorption,detergency and antistatic properties

The adsorption of a mixture of cationic (c) and anionic (a) surfactants on cellulose fibers is highly dependent on the molar ratio a/c with a maximum at a/c = 0.9. When a/c is > 1.1 the adsorption is negligible. The presence of nonionic surfactants in the solution impairs the adsorption of the ionic species; this effect is stronger for nonionic surfactants with long alkyl and polyglycol ether chains. The detergency—measured on WFK cotton cloth—is highest when a/c > 1 and decreases sharply when a/c goes below 0.8. The antistatic effect for a formulated liquid detergent based on these principles was compared to one commercial liquid detergent with softening and antistatic properties and one commercial detergent powder, and the test detergent was shown to be a better antistatic agent on polyester, polyacrylonitrile and polyamide. The detergency was about the same for the two liquid detergents.     

Two soybean genotypes lacking lipoxygenase-1

The U.S. Department of Agriculture soybean germplasm collection (6,499 accessions) was screened for genotypes with greatly reduced or missing lipoxygenase-1 (L-1) [linoleate: O₂ oxidoreductase, EC 1.13.11.12] and lipoxygenase-2 and L-3 (L-2 and L-3) activity. The L-1 assay used linoleic acid dispersed in Tween-20 at pH 9.0 as the substrate (acid assay) and the L-2 and L-3 assay used linoleic acid methyl ester dispersed in ethanol at pH 7.0 as the substrate (ester assay). The spectrophotometric assay based on conjugated diene formation at 234 mm was used in the qualitative screening procedure. Two plant introductions (PI), 133226 from Indonesia and PI 408251 from Korea lacked L-1 activity. Oxygen uptake, electrophoresis and isoelectric focusing confirm the lack of detectable L-1 activity in the seed of these two genotypes. Radial diffusion against soybean seed lipoxygenase antiserum showed that the two genotypes are missing a precipitin band that normal soybean genotypes and purified lipoxygenase from soybean seed exhibit. Neither the L-1 variants nor any other accessions tested had greatly reduced activity with the ester assay. An erratum to this article is available at .     

Partial purification and characterization of a Ca2+-stimulated lipoxygenase from soybean seeds

A highly purified Ca²⁺-stimulated lipoxygenase was isolated from the Hill variety of soybean seeds. Separation of Ca²⁺-stimulated lipoxygenase from lipoxygenase active in the absence of Ca²⁺ (lipoxygenase-1) was readily obtained using a DEAE-cellulose column. Sample size applied to the ion exchange column was found to be critical. Both enzymes were bound to the column, although some highly active Ca²⁺-stimulated lipoxygenase eluted with buffer in the presence of bound lipoxygenase-1. Ca²⁺-stimulated lipoxygenase bound to DAAE-cellulose required the use of a NaCl gradient for elution. Ca²⁺-stimulated lipoxygenase showed an apparent isoelectric point at pH 5.90 and optimum activity at pH 7.5 and at 1.1 mM calcium. Lipoxygenase-1 was inhibited over 95% in the presence of 60 μM methyl mercuric chloride, while Ca²⁺-stimulated lipoxygenase showed a maximum of only 20% inhibition under the same conditions. Paper No. 4993 Mississippi Agricultural and Forestry Experiment Station.     

Biosurfactants from Urban Wastes for Detergent Formulation: Surface Activity and Washing Performance

To promote biobased products for industry, six biosurfactants (BS) isolated from green and food urban residues aged under aerobic digestion for 0–60 days were investigated for their chemical composition, surface activity properties and detergent performance in fabric washing in comparison with commercial anionic and nonionic surfactants (CS). The BS exhibited a range of chemical composition and surface activity properties presumably related to their different biomass sources. Surface tension data for these substances correlated meaningfully with performance data in fabric washing. It was found that BS have similar performances as CS when used neat, but the 1:1 w/w BS–CS mixes are characterized by significant synergy. High sensitivity to water hardness and fabric yellowing were found to be the main deficiencies for the BS. However, both effects are minimized or are not critically evident when the BS are used together with CS and/or in common detergents formulations. The results indicate that when used above critical micellar concentration, no significant or critical performance differences are evident within the whole group of BS or between BS and CS. These facts encourage expectations for industrial production and real commercialization of BS as components of detergents formulations.     

Establishment of a model substrate oil for antioxidant activity assessment by oil stability index method

A model substrate oil using methyl linoleate was established for the determination of the antioxidant activity by Oil Stability Index (OSI) method. OSI values for methyl linoleate with different concentrations (5–100%) in silicone oil were measured at different temperatures (70–120°C). As the temperature increased, the OSI value decreased in each concentration of methyl linoleate. Optimal temperature and concentration of antioxidants, α-tocopherol, and butylated hydroxytoluene on OSI values for 10% methyl linoleate model oil was measured at 90, 100, 110, and 120°C. The logarithmic relationship between temperature and OSI using model substrate oil was similar to that of soybean oil. Furthermore, application of some spice extracts to this model oil system was carried out to give results thhat compared well with those available in the literature. Thus, the procedure using methyl linoleate-silicone oil as a model substrate oil is available for evaluating the antioxidant activity by the OSI method.     

Inhibition of lipoxygenase 1 by phosphatidylcholine micelles-bound curcumin

Curcumin (diferuloyl methane) from rhizomes of Curcuma longa L. binds to phosphatidylcholine (PC) micelles. The binding of curcumin with PC micelles was followed by fluorescence measurements. Curcumin emits at 490 nm with an excitation wavelength of 451 nm after binding to PC-mixed micelles stabilized with deoxycholate. Curcumin in aqueous solution does not inhibit dioxygenation of fatty acids by Lipoxygenase 1 (LOX1). But, when bound to PC micelles, it inhibits the oxidation of fatty acids. The present study has shown that 8.6 μM of curcumin bound to the PC micelles is required for 50% inhibition of linoleic acid peroxidation. Lineweaver-Burk plot analysis has indicated that curcumin is a competitive inhibitor of LOX1 with K _l of 1.7 μM for linoleic and 4.3 μM for arachidonic acids, respectively. Based on spectroscopic measurements, we conclude that the inhibition of LOX1 activity by curcumin can be due to binding to active center iron and curcumin after binding to the PC micelles acts as an inhibitor of LOX1.     

Importance of micellar relaxation time on detergent properties

As we enter the new millennium, manufacturers of laundry detergents would like to provide new products for the twenty-first century. With the goal of achieving new and better performance characteristics, design strategies for research and development should be defined. This paper highlights the importance of micellar relaxation kinetics in processes involved in detergency. Earlier Shah and coworkers showed that the stability of sodium dodecyl sulfate (SDS) micelles plays an important role in various technological processes. The slow relaxation time (τ₂) of SDS micelles, as measured by the pressure-jump technique, was in the range of 10⁻⁴ to 10¹ s, depending on the surfactant concentration. A maximal relaxation time and thus a maximal micellar stability was found at 200 mM SDS (5 s), corresponding to the least-foaming, largest bubble size, longest wetting time of textile, largest emulsion droplet size, and the most rapid solubilization of oil. These results are explained in terms of the flux of surfactant monomers from the bulk to the interface, which determines the dynamic surface tension. More stable micelles lead to less monomer flux and hence to a higher dynamic surface tension. The relaxation time for nonionic surfactants (as measured by the stopped-flow technique) was much longer than for ionic surfactants because of the absence of ionic repulsion between the head groups. The τ₂ was related to dynamic surface-tension experiments. Stability of SDS micelles can be greatly enhanced by the addition of long-chain alcohols or cationic surfactants. In summary, relaxation time data of surfactant solutions enable us to predict the performance of a given surfactant solution. Moreover, results suggest that one can design appropriate micelles with specific stability, or τ₂, by controlling surfactant structure, concentration, and physicochemical conditions, as well as by mixing anionic/cationic or ionic/nonionic surfactants for a desired technological application, e.g., detergency.     

Determination of the phospholipase activity of patatin by a continuous spectrophotometric assay

Patatin is a family of glycoproteins that accounts for 30–40% of the total soluble protein in potato (Solanum tuberosum L.) tubers. This protein has been reported to serve as a storage protein and also to exhibit lipid phospholipase activity. This paper describes a simple continuous spectrophotometric method for assaying patatin phospholipase activity. The procedure is based on a coupled enzymatic assay using [1,2-dilinoleoyl]PC as the phospholipase substrate and lipoxygenase as the coupling enzyme. In the procedure developed in this work, lipoxygenase oxidizes the linoleic acid released by the phospholipase activity of patatin. This activity can then be followed spectrophotometrically by recording the increase in absorbance at 234 nm that results from the formation of the corresponding hydroperoxide from linoleic acid by the action of lipoxygenase. The optimal assay concentrations of patatin and lipoxygenase were established. Phospholipase activity varied with pH, reaching its optimal value at pH 9.5. Scans of the deoxycholate concentration pointed to an optimal detergent concentration of 3 mM. Phospholipid hydrolysis followed classical Michaelis-Menten kinetics (V _m=9.8×10⁻³ μmol/min·μg protein, K _m=7.8 μM, V _m/K _m=1.3 min⁻¹·μg protein). This method proved to be specific since there was no activity in the absence of patatin. It also had the advantages of a short analysis time and the use of commercially nonradiolabeled and inexpensive substrates, which are, furthermore, natural substrates of phospholipase.     

Mechanism of lower oxidizability of eicosapentaenoate than linoleate in aqueous micelles

The aerobic oxidation kinetics of methyl eicosapentaenoate (20:5n-3) and methyl linoleate (18:2n-6) were compared in homogeneous chlorobenzene solution and in Triton X-100 aqueous micelles at 37°C. The rate of disappearance of 20:5n-3 was two times faster than that of 18:2n-6 in chlorobenzene, while the former was five times slower than the latter in aqueous micelles. It was also observed that ΔO₂=Δ18:2n-6 and ΔO₂=2Δ20:5n-3 in aqueous micelles. In the oxidation of a 1∶1 mixture of 20:5n-3 and 18:2n-6 in micelles, the rate of disappearance of 20:5n-3 was 3.6 times faster than that of 18:2n-6, and the rate of total substrate disappearance was reduced by a factor of 5 as compared with 18:2n-6 oxidation. These data suggest that the peroxyl radical derived from 20:5n-3 is more polar than that from 18:2n-6, and the former is likely to diffuse from the core to the micelle surface. This lowers the oxidizability for 20:5n-3 in aqueous micelles by enhancing the termination reaction rate for peroxyl radicals and by reducing the rate of propagation since there may be more 20:5n-3 peroxyl radicals at the surface than in the micelle core.     

Characterization of phospholipase A2 from rabbit lung microsomes

A phospholipase A₂ activity associated with the microsomal fraction of rabbit lung homogenates was studied. The enzyme showed specificity for thesn ⁻² ester bond of phosphatidylcholine, had an alkaline pH optimum and required Ca²⁺ for activity. Other divalent cations were unable to support hydrolysis. In the absence of detergents, exogenous phosphatidylethanolamine was deacylated at a rate sevenfold higher than phosphatidylcholine. The activity toward both substrates could be enhanced by sodium deoxycholate or, more effectively, by sodium taurodeoxycholate. Phosphatidylethanolamine required higher detergent/phospholipid molar ratios than phosphatidylcholine. Under these conditions, the preference for the former substrate over the latter was nearly abolished. The zwitterionic detergent 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) and the nonionic detergent Triton X-100 were either ineffective (phosphatidylcholine) or inhibitory (phosphatidylethanolamine). Addition of KCl produced opposite effects on the activity depending on the bile salt used to disperse the substrate. The phospholipase A₂ activity was inhibited byp-bromophenacyl bromide but remained unaffected after treatment with diisopropylfluorophosphate or NaF. N-Ethylmaleimide, but not other thiol reagents, partially inhibited the activity. Presented in part at the 29th CFBS meeting, Guelph, Canada, June 1986, and at the symposium “25 Years Lipids and Biomembranes,” Utrecht, The Netherlands, June 1986.     

Kinetics of hydrogenation of conjugated triene and diene with nickel,palladium, platinum and copper-chromite catalysts

A mixture of methyl linoleate and alkali-conjugated methyl linoleate was reduced with nickel, palladium, platinum and copper-chromite catalysts. The course of hydrogenation was followed by gas liquid chromatography of samples withdrawn at intervals. Relative rate constants of reactants and inermediates were calculated by a computer. Conjugated linoleate was 10–18 times more reactive than methyl linoleate with all catalysts except platinum, which showed no selectivity at 60 C. At 150 C conjugated diene reacted four times faster than methyl linoleate with platinum catalyst. A conjugated diene-to-stearate shunt was observed with palladium and platinum catalysts. When β-eleostearate was hydrogenated with the same catalysts, 50–97% of the triene was reduced directly to monoene with all catalysts except copper chromite, which selectively reduced conjugated triene to conjugated diene. On the basis of present kinetic data and previous knowledge about the mode of hydrogen addition to conjugated systems, a scheme has been proposed to account for the products formed during hydrogenation of methyl linolenate. ARS, USDA.     

Production of 9-hydroperoxy-γ-linolenic acid by soybean lipoxygenase in a two-phase system

9-Hydroperoxy-γ-linolenic acid (9-GOOH) was produced selectively by soybean lipoxygenase (LG) from γ-linolenic acid (GLA) using a two-phase (borate buffer, pH 6.5/hexane) system at a low temperature (10°C) with some anionic surfactants that showed little inhibitory effect on the enzyme at pH 6.5. The system avoided the inhibitory effect of a higher substrate concentration and of hydroperoxide as well as low substrate solubility in an aqueous system. Not lipoxygenase-2 (LG2) but lipoxygenase-1 (LG1) was indicated to be responsible for the production of 9-GOOH. Among the anionic surfactants examined, acetate and sarcosinate were shown to be suitable, but phosphate was not. Ca²⁺ increased the 9-GOOH productivity. The LG1 fraction gave the maximum yield of 35% with 0.5 mM Ca²⁺, ECT-3N (anionic surfactant, acetate) at 10°C and at 4.8 mM GLA in an emulsion.     

Inhibition of soybean lipoxygenase-1 by chain-breaking antioxidants

The aim of this investigation was to determine whether chain-breaking antioxidants able to prevent lipid peroxidation can inhibit lipoxygenase-1 (EC 1.13.11.12). Therefore, the effects of ascorbic acid, 6-palmitoylascorbic acid and trolox on the enzyme activity were analyzed by means of Lineweaver-Burk double reciprocal plots and Yoshino's graphical method. The effect of these compounds on the formation of free radicals during lipoxygenase-1 reaction was investigated as well, by monitoring the enzymic formation of oxodienes. We present evidence that the chain-breaking antioxidants ascorbic acid, 6-palmitoylascorbic acid and trolox inhibit soybean lipoxygenase-1 in the micromolar concentration range (Ki 27, 3 and 18 μM, respectively). The inhibition is competitive, complete and reversible. All three compounds trap the free radicals formed during the lipoxygenase-catalyzed reaction, which might substantially contribute to their inhibitory ability. These findings can have physiological significance in the light of the lipoxygenase involvement in biomembrane remodelling.     

Effect ofd-α-tocopherol analogues on lipoxygenase-dependent peroxidation of phospholipid-bile salt micelles

In order to know whether or not vitamin E acts as an effective antioxidant in lipoxygenase-dependent peroxidation of phospholipids, the effect of vitamin E and vitamin E analogues, 2,2,5,7,8-pentamethyl-6-hydrohychroman (PMC) and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox C), was investigated in enzymatic lipid peroxidation of bile salt micelles of pig liver phosphatidylcholine (PC) using soybean lipoxygenase. 15-Hydroperoxy-5,8,11,13-eicosatetraenoic acid was exlusively produced by the reaction with the PC molecular species containing arachidonic acid moiety, indicating that the hydroperoxidation of pig liver PC entirely progresses through the enzymatic reaction. PMC suppressed the accumulation of PC-hydroperoxides (PC-OOH) more efficiently than eitherd-α-tocopherol (α-Toc) or Trolox C, and 50% inhibition concentration by PMC was close to that of quercetin, a known lipoxygenase inhibitor from natural origin. The antioxidant activity of PMC was also superior to that of either α-Toc or Trolox C in ferrous ion-induced nonenzymatic oxidation of PC micelles in the presence of a trace amount of PC-OOH, although the radical-scavenging activities of these compounds in solution were similar or comparable to one another. In conclusion, PMC is more effective than α-Toc as an inhibitor of lipoxygenase reaction with phospholipids and of autoxidation in phospholipids. The phytyl chain of α-Toc seems to be unfavorable for exerting an inhibitory effect on lipoxygenase reaction with phospholipid-bile salt micelles.     

Detergency measurement using artificially-soiled cloths

Summary and Conclusions Four types of artificially-soiled cloths are compared in their ability to evaluate cleaning of representative detergent products. Three of these cloths are commercially available while the fourth is from a private laboratory. These are compared in their ability to rate detergents in the same order as naturally-soiled clothes rate detergents. Sensitivity and reproducibility of the various soiled cloths in measurement of soil removal and whiteness retention are studied. Results show that artificially-soiled cloths must be used advisedly. There is no substitute for actual performance tests of detergent products under practical conditions. At best, artificially-soiled cloths are useful for “screening” purposes where positive test results are confirmed by practical tests. Presented at the 25th annual fall meeting, American Oil Chemists’ Society, Chicago, Ill., October 8–10, 1951.