Simo Laakso

Bile salt-dependent oxygenation of polyunsaturated phosphatidylcholines by soybean lipoxygenase-1

Abstract Aqueous solutions of purified bile salts and phosphatidylcholine (PC) were susceptible to oxygenation by soybean lipoxygenase-1. The bile salts could not be replaced by e.g., Tween 20 or 80, octylglucoside, Triton X-100, SDS or sulphobetaine based ampholytic detergents. The liberation of the fatty acid moieties of PC prior to oxygenation did not occur. The rate of oxygenation showed complex dependency on the concentration of bile salts whereas a linear increment with increasing PC concentrations could be demonstrated by using constant bile salt: fatty acid ratios. We expected, therefore, that a specific bile salt-PC complex instead of monomer phospholipid was required in the catalysis by lipoxygenase-1. Ultraviolet spectroscopic and luminometric monitoring of PC oxygenation supported this view: (i) The formation of conjugated dienes corresponded to only about 50% of the O 2 consumed as calculated on molar basis, (ii) Total absence of luminol chemiluminescence suggested that the other half of the consumed oxygen was not utilized to luminol oxidation, but either caused the cooxidation of bile salts in a coupled reaction with the oxygenation of PC or was incorporated into nonconjugated double bond structures of PC. Special attention was called to the applicability of soybean lipoxy genase-1 in the preparation of peroxidized phospholipid liposomes.

Colorimetric determination of β-cyclodextrin: two assay modifications based on molecular complexation of phenolphatalein

The decolorization of phenolphtalein upon complexation to cyclodextrins was studied to measure beta-cyclodextrin concentrations. Several factors possibly affecting the self-life of the dye were tested. By making the assays in 0.1 M NaCO3 solution beta-cyclodextrin concentrations down to 6 microM (SNR = 2) could be determined while the practical assay range reached up to 0.06 mM. In this form the method was unaffected by acyclic oligosaccharides and directly applicable to cyclodextrin assays in complex starch hydrolyzates. The method was further modified to be used in a flow-injection analyzator and the results were comparable to those obtained by HPLC analyses after prepurification of the samples.

Inhibition of lipid peroxidation by casein Evidence of molecular encapsulation of 1,4-pentadiene fatty acids

The capability of cyclodextrins to form molecular inclusion complexes with linoleate appeared in a lipoxygenase-linoleate model reaction as inhibition of oxygenation. The inhibited rates were established instantaneously upon addition of the complexant and maintained until linoleate was exhausted. Total cessation of the reaction was not obtained with cyclodextrins. All these features were reproduced also in casein-inhibited reaction mixtures. Both casein and cyclodextrins protected linoleate also against autoxidation although they did not change free radical generation by xanthine oxidase or Fe2+ reactions. Since neither of the inhibitors affected the enzyme directly, casein may also act by forming linoleate complexes which via a standing equilibrium reduce the oxidizable monomer fatty acids and cause substrate-limited reaction rates. Comparisons at acidic and alkaline pH, in the presence of increasing amounts of the complexants, detergent and hydroperoxides supported this view.

A sensitive lipoxygenase assay based on chemiluminescence

Abstract The presence of luminol during lipoxygenase I-catalyzed oxygenation of linoleic acid caused a complex pattern of emission of chemiluminescent light. During the aerobic phase of the lipoxygenase reaction, the rate of formation of light was dependent on the enzyme, linoleic acid, and luminol concentrations. The correlation was linear between the rate of chemiluminescent light formation and lipoxygenase I concentration in the range 2–22 mU/ml of enzyme in the presence of saturation concentrations of linoleic acid (0.4 m m ) and luminol (0.04 m m ). Luminol and derivatives of isoluminol were all applicable as chemiluminescent probes without an inhibitory effect on the oxygenation rate itself. Free radicals that dissociated from the enzyme-substrate complex in the presence of oxygen in a constant ratio to the oxygenation rate are suggested to be responsible for luminol oxidation.

A spectrophotometric assay for demethiolating activity

Abstract A rapid method for detection of demethiolating activity in bacterial suspensions is described. The procedure is based on spectrophotometric determination of aryl mercaptan produced by quantitative reaction of 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) with liberating methanethiol. Under the conditions used (30°C, pH 7.4) the reduction of DTNB by methanethiol was linear over 20 min. A linear correlation between demethiolation rate and cell concentration was observed up to 0.5 mg dry wt/ml. DTNB had no effect on methionine transport or normal growth of Pseudomonas fluorescens UK-1.

Immobilized lipoxygenase in continuous production of fatty acid hydroperoxides

Soybean lipoxygenase-1 was covalently coupled to agarose with 75% recovery of catalytic activity. Because evidence was obtained that the immobilization resulted in improved operational stability of the enzyme, a lipoxygenase-reactor and a continuous process for the synthesis of 13-hydroperoxy-linoleic acid and 15-hydroperoxyarachidonic acid were developed. A procedure based on spectrophotometric hydroperoxide assay and constant oxygraphic monitoring of the effluent is presented for the calibration of the reactor to operate at the highest conversion efficiency when oxygenating quantitatively the substrate. Under these conditions, the reactor was capable of producing about 0.6 mg of hydroperoxy fatty acid/1.0 ml of wet gel/hr. The covalently coupled enzyme has been stable during six months of storage at 3 C in 0.2 M Na-borate buffer, pH 9.0, and during the same period, its operational stability in the column has been unaltered under the conditions used.

[14] Assays of lipoxygenase, 1,4-pentadiene fatty acids, and O2 concentrations: Chemiluminescence methods

Publisher Summary Lipoxygenases are iron-containing enzymes that catalyze dioxygenation, by molecular oxygen, of cis,cis-1,4-pentadiene fatty acids. The reaction is a source of weak chemiluminescence due to the dissociation of free radicals from the main path of hydroperoxidation. Under alkaline conditions, the intensity of light emission can be drastically amplified by the presence of luminol. The kinetics of luminol chemiluminescence in the reaction catalyzed by soybean lipoxygenase-I is coupled to the changes in O 2 concentration. In air-saturated reaction mixtures, the initial oxygenation phase emits light in proportion to the rate of the overall process measured by O 2 consumption or by the accumulation of the conjugated diene products. When dissolved O 2 is consumed to a certain critical level, a second emission peak appears. This has been taken to indicate the increased dissociation of enzyme-bound oxygen or fatty acid free radicals, which react with the remaining oxygen prior to the initiation of the chemiluminescence-silent anaerobic catalysis. Consequently, the emission curve is two-phasic whenever the molar concentration of the substrate fatty acid exceeds that of dissolved O 2 . The former phase forms the basis for luminometric lipoxygenase assays, whereas the latter serves to assay O 2 concentrations in samples added to the anaerobic mixtures of lipoxygenase-1 and linoleic acid. The luminometric procedures described offer high sensitivity combined with the ease of performance.

Determination of cis,cis-methylene interrupted polyunsaturated fatty acids in aqueous solutions by lipoxygenase chemiluminescence

The chemiluminescent reaction of luminol during lipoxygenase-catalyzed oxygenations was studied with the purpose of developing a specific luminometric assay for cis,cis-1,4-pentadiene fatty acids directly in aqueous solutions. The addition of picomole levels of either linoleic or arachidonic acids to reaction systems containing 0.04 mM luminol and 40 micrograms/ml of purified soybean lipoxygenase-1 gave light emission curves with a single sharp maximum. Under these conditions the peak heights were linearly dependent on the fatty acid concentration and the detection limit for both of the fatty acids was 2 pmol with a signal to noise ratio of 2. For maximum reproducibility of the assays a procedure for the proper quantitation of the enzyme was developed. The fact that the assay proved to be relatively interference-free was ascribed to the high molar enzyme/substrate ratio (above 1).

A microassay of O2 concentration based on oxygen-induced chemiluminescence in anaerobic lipoxygenase reactions

The preparation of chemiluminescence probes for assaying O2 concentrations in microsamples is described. This probe is based on an anaerobic lipoxygenase-linoleate system continuously generating reactive intermediates which in a spontaneous reaction with added O2 yield an excited species. The resulting chemiluminescence signals are highly reproducible upon repeated sample application and unaffected by even large variations in the contents of lipoxygenase-1 and linoleic acid. The linear assay range is between 0.25 and 25 nmol of O2. The assay system described is stable for 90 +/- 10 min, irrespective of the number of samples added, and the probe can be regenerated thereafter by adding linoleic acid.

Altered regulation of macromolecular synthesis in methionine-inhibited cultures of Pseudomonas fluorescens UK1.

Effects of high methionine concentrations on growth of Pseudomonas fluorescens UK1 are reported. The following phenomena were observed: (i) Immediate inhibition of growth for a period corresponding to approximately half a generation. Steady-state conditions of growth were no more attained. (ii) In spite of stringency of the macromolecular synthesis in this organism, simultaneously with the growth inhibition, the rate of labelled leucine incorporation into trichloroacetic acid (TCA)-insoluble material was reduced 60% while the rate of labelled uracil incorporation remained constant. (iii) The organism began to liberate methanethiol half a generation after the methionine supplement. Demethiolating activity increased linearly with the cell mass. It is concluded that the inhibition of growth is not due to the liberation of methanethiol from methionine but the amino acid is able to uncouple the mechanism coordinating protein and RNA synthesis in P. fluorescens UK1.