Speciation and subcellular location of Se-containing proteins in human liver studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and hydride generation-atomic fluorescence spectrometric detection

Speciation of Se-containing proteins in the subcellular fractions of human liver was studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by hydride generation-atomic fluorescence spectrometric (HG-AFS) detection. It was found that about 24 kinds of Se-containing proteins existed in subcellular fractions of normal human liver. The molecular weights (MW) of the subunits were mostly in the range 20-30 kDa and 50-80 kDa. Major Se-containing protein fractions at 61 kDa and 21 kDa are probably selenoprotein P and glutathione peroxidase, respectively. The 54 kDa protein is probably a thioredoxin reductase, which is presented in nuclei, mitochondria, lysosome, microsome and cytosol. We noticed that the Se-containing protein with the lowest MW of 9.3 kDa only existed in lysosome. Most of the proteins have not been identified and would require further investigation to characterize them. The specific subcellular distributions of different Se-containing proteins suggest that they could play important biological roles in each organelle.     

Molecular mass determination of plasma-derived glycoproteins by ultraviolet matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with internal calibration

Human plasma-derived antithrombin III (AT-III), factor IX (FIX) and vitronectin (VN) were characterized as native glycoproteins and in their de-N-glycosylated form by means of MALDI mass spectrometry. The average molecular masses of the three complex glycoproteins were determined applying internal calibration with high-mass, well-defined protein calibrants. Internal calibration generated for the 47 kDa yeast protein enolase a mass precision in the continuous and delayed extraction mode of +/-0.12 and +/-0.022%, respectively. The achievable mass accuracy for such a high-mass, unmodified protein was in the range of 0.02% in the continuous mode, which turned out to be better than in the delayed extraction mode. Purification of all (glyco) proteins (even the calibration proteins) by means of ZipTip technology and direct elution with a solvent system containing the appropriate MALDI matrix turned out to be a prerequisite to measure the exact molecular masses with an internal calibration. The average molecular masses of the two different forms of AT-III, namely AT-III(alpha) and AT-III(beta), were shown to be 57.26 and 55.04 kDa, respectively. The 2.22 kDa mass difference is attributed to the known difference in carbohydrate content at one specific site (Asn-135). After exhaustive de-N-glycosylation (by means of PNGase F) of the alpha- and beta-form and subsequent MALDI-MS analysis, average molecular masses of 48.96 and 48.97 kDa, respectively, were obtained. These values are in good agreement (-0.15%) with the calculated molecular mass (49.039 kDa) of the protein part based on SwissProt data. The molecular mass of the heavily post-translational modified glycoprotein FIX was found to be 53.75 kDa with a peak width at 10% peak height of 4.5 kDa, because of the presence of many different posttranslational modifications (N- and O-glycosylation at multiple sites, sulfation, phosphorylation, hydroxylation and numerous gamma-carboxyglutamic acids). MALDI-MS molecular mass determination of the native, size-exclusion chromatography-purified, VN sample revealed that the glycoprotein was present as dimer with molecular mass of 117.74 kDa, which could be corroborated by non-reducing SDS-PAGE. After sample treatment with guanidine hydrochloride and mass spectrometric analysis, a single, new main component was detected. The molecular mass turned out to be 59.45 kDa, representing the monomeric form of VN, known as V75. The determined molecular mass value was shown to be on one hand lower than from SDS-PAGE and on the other higher than the calculated amino acid sequence molecular mass (52 277 Da), pointing to the well-known SDS-PAGE bias and to considerable post-translational modifications. Further treatment of the sample with a reducing agent and subsequent MALDI-MS revealed two new components with molecular masses of 49.85 and 9.41 kDa, corresponding to V65 and V10 subunits of VN. PNGase F digest of the V75 and V65 units and MS analysis, exhibiting a molecular mass reduction of 6.37 kDa in both cases, verified the presence of a considerable amount of N-glycans.     

Simultaneous separation of anionic and cationic proteins by capillary electrophoresis using high concentration of poly(diallyldimethylammonium chloride) as an additive

The simultaneous separation of anionic and cationic proteins has been achieved by addition of high concentration of poly(diallyldimethylammonium chloride) (PDDAC) in capillary electrophoresis. A capillary was filled with PDDAC so that it would act as ion-pair reagents in the separation of anionic proteins. On the other hand, the PDDAC can also be used as coating additives for the analysis of cationic proteins. Increasing the concentration of PDDAC in the separation buffer had the ability to improve the separation efficiency, change the electrophoretic mobility, and alter the separation selectivity; however, this was not true in the case of analyzing proteins by using the PDDAC larger than 1.6%. By both using a buffer containing 1.6% PDDAC and applying pH-stepwise techniques, 13 proteins with a wide range of pI (4.7-11.1) and molecular masses (6.5-198.0 kDa) could be separated within 30 min in a single run. In addition to this separation, we observed not only more peaks from alpha-chymotrypsinogen A and aprotinin but also the bovine serum albumin (BSA) dimer and trimer. With the 50 nL protein injection sample, the limits of detections at signal-to-noise of 3 for proteins are in the range of 0.07-0.79 microM. Except for BSA, the relative standard derivation values of migration time and peak height for all proteins were <1.3 and <6.9%, respectively. We suggested that this proposed method is a promising approach for clinical diagnosis and proteomics applications.     

Towards the characterization of metal binding proteins in metal enriched yeast

This paper presents size exclusion chromatography data with on-line coupling to UV and inductively coupled plasma mass spectrometry (ICP-MS) of water soluble metal-binding compounds present in zinc, copper, chromium and iodine enriched yeast nutritional supplements. Molecular weight estimates of the extracted metal-containing compounds are given and are shown to vary substantially from 1.2 kDa to larger than 668 kDa. Seven proteins suspected of containing chromium were identified from one of the chromium-containing fractions. Four of these identified proteins are known to form complexes with other metal ions. The metal chromatographic profiles of zinc, copper and chromium-enriched yeasts were compared to their respective native metal profiles in non-enriched yeast samples. The chromium profiles are shown to be markedly different while those of zinc and copper are qualitatively similar. Only iodide ions or weakly bound, non-aromatic, low molecular weight ( 1.2 kDa) iodine species were observed in the iodine-enriched yeast samples.     

Study of Chromium Speciation in Normal and Diabetic Rats by Activable Enriched Stable Isotope Technique

Chromium speciation was investigated in the liver cytosol, serum and urine of normal and diabetic rats after a single intravenous injection of enriched stable isotope ⁵⁰Cr tracer solution. Sephadex G-25 gel chromatography combined with instrumental neutron activation analysis was used to isolate and characterize protein-bound chromium in the above materials. The results indicate that Cr is mainly combined with a high-molecular-weight protein either in liver cytosol or serum. A low-molecular-weight, Cr-containing compound (LMWCr) was found in all the observed liver, serum and urine samples of both normal and diabetic rats. Chromium is excreted chiefly as LMWCr in urine.     

Monitoring of water content and water distribution in ischemic hearts

We determined water content and water distribution by fitting dielectric spectra of ischemic canine hearts between 5 MHz and 3 GHz with a newly developed model which describes heart cells and subcellular organelles as rotational ellipsoids filled with electrolyte enclosed by an isolating membrane. The fraction of dry material is modelled by spherical particles with a small dielectric permittivity. Free model parameters were water content, cell volume fraction, and the conductivity of the electrolytes. Resulting model parameters were compared to data from tissue desiccation and to conductivity changes produced by protons and lactate ions. We investigated hearts in two states: during ischemia after interruption of blood flow (pure ischemia, PI, n=5) and during ischemia after resuscitation with Tyrode's solution (IAR, n=14).The difference between water content determined by tissue desiccation and by dielectric spectroscopy was less than 0.5%. During 360 min of ischemia, water content in IAR decreased from 85+/-1.6% to 83+/-2.2% and in PI from 80+/-0.8% to 78+/-1.5%. Cellular volume fraction in IAR increased from 0.47+/-0.045 to 0.63+/-0.031 and in PI from 0.62+/-0.014 to 0.73+/-0.013, which is consistent with published morphometric data. After 180 min of ischemia, the increase of the cytosolic conductivity was 0.14+/-0.02 S/m as calculated from the dielectric spectrum and was similar to the conductivity increase which was roughly estimated on the basis of tissue lactate concentration.In conclusion, dielectric spectroscopy combined with our model analysis facilitates the monitoring of water content and distribution by means of nondestructive surface probes.     

Towards the proteome of the rhodopsin-bearing post-Golgi compartment of retinal photoreceptor cells

Polarized sorting of rhodopsin in retinal rod photoreceptor cells is mediated by post-Golgi carrier membranes that bud from the trans-Golgi network and fuse with the specialized domain of the plasma membrane in the rod inner segment. The identity of the majority of the resident proteins of this organelle still remains elusive, despite multifaceted approaches to study this compartment. In the present study we have taken a proteomic approach to the analysis of the post-Golgi carriers. First, we modified the previously established fractionation protocols in order to achieve greater purity of the isolated membranes. Specifically, the new fractionation scheme depleted the post-Golgi fraction of cytosolic proteins that were the most abundant contaminants complicating analysis of two-dimensional (2-D) gel profiles in our previous preparations. The isolated membranes were subjected to 2-D gel electrophoresis, immunoblotting and microsequencing. This analysis showed that the improved subcellular fractionation yielded a fraction highly enriched in rhodopsin-bearing post-Golgi carrier membranes. Two-dimensional mapping revealed 29 proteins that are preferentially found in this fraction and therefore represent candidates for post-Golgi membrane-specific proteins. This preparation of rhodopsin-bearing post-Golgi carriers is a first step towards the proteomics of this important organelle.     

High-efficiency protein extraction from polyacrylamide gels for molecular mass measurement by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry

A simple and fast method of protein extraction from Coomassie Brilliant Blue (CBB)-stained polyacrylamide gels suited for molecular mass measurement of proteins by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) is reported. Proteins in CBB-stained gel pieces were extracted by a 10-min soaking in 0.1 M NaOH at 25 degrees C. The recovery of this one-step extraction method was 34-73% for proteins <67 kDa. CBB adduction to proteins during mass spectrometric analysis was avoided by a destaining step before the alkaline extraction. The molecular mass values of the extracted proteins coincided with those of purified proteins within +/-0.01-0.10% deviation for all the proteins <36 kDa. Because of the high extraction recovery, mass measurement was possible for the proteins extracted from CBB-stained gels with loaded protein quantities as little as 34 ng for cytochrome c, alpha-lactalbumin, myoglobin, beta-lactoglobulin, trypsinogen, and carbonic anhydrase (12.4-29.0 kDa), 340 ng for glyceraldehyde-3-phosphate dehydrogenase (35.6 kDa) and albumin (66.3 kDa). This method provides a highly efficient approach to utilize CBB-stained one- or two-dimensional gels for whole protein analysis using MALDI-TOF-MS.     

X-ray absorption and EPR spectroscopic studies of the biotransformations of chromium(VI) in mammalian cells. Is chromodulin an artifact of isolation methods?

Very different biological activities are usually ascribed to Cr(VI) (a toxin and carcinogen) and Cr(III) (an antidiabetic agent), although recent evidence suggests that both these types of actions are likely to arise from cellular uptake of varying concentrations of Cr(VI). The first systematic study of XANES spectra of Cr(III) complexes formed in Cr(VI)-treated mammalian cells (A549, HepG2, V79, and C2C12 cell lines), and in subcellular fractions of A549 cells, has been performed using a library of XANES spectra of model Cr(III) complexes. The results of multiple linear regression analyses of XANES spectra, in combination with multiple-scattering fits of XAFS spectra, indicate that Cr(III) formed in Cr(VI)-treated cells is most likely to bind to carboxylato, amine, and imidazole residues of amino acids, and to a lesser extent to hydroxo or aqua ligands. A combination of XANES and EPR spectroscopic data for Cr(VI)-treated cells indicates that the main component of Cr(III) formed in such cells is bound to high-molecular-mass ligands (>30 kDa, probably proteins), but significant redistribution of Cr(III) occurs during the cell lysis, which leads to the formation of a low-molecular-mass (<30 kDa) Cr(III)-containing fraction. The spectroscopic (XANES, XAFS, and EPR) properties of this fraction were strikingly similar to those of the purported natural Cr(III)-containing factor, chromodulin, that was reported to be isolated from the reaction of Cr(VI) with liver. These data support the hypothesis that a chromodulin-like species, which is formed from such a reaction, is an artifact of the reported isolation procedure.     

Retention of Large Biological Molecules by Size-Exclusion Chromatography

Size-exclusion chromatography has been developed for the separation of large biological molecules including proteins, polymers, peptides, nucleic acids, and polysaccharide according to their molecular size. This study determined the retention factors for dextran (5, 25, 50, 270, 670, and 1100?kDa) and polysaccharides, such as fucoidan, alginic acid, and laminarin, in the size-exclusion chromatography stationary phase. In addition, the molecular weights and retention factor of three polysaccharides were calculated from the dextran standard curve. The largest retention factor was 4.26 using the size-exclusion chromatography columns (5?kDa dextran). The molecular weights of fucoidan, alginic acid, and laminarin were determined to be 250, 200, and 5 to 64?kDa, respectively.     

Isoelectric focusing nonporous silica reversed-phase high-performance liquid chromatography/electrospray ionization time-of-flight mass spectrometry: a three-dimensional liquid-phase protein separation method as applied to the human erythroleukemia cell-line

A liquid-phase three-dimensional protein separation method has been developed that is used to separate the cytosolic fraction of a HEL cell lysate via isoelectric focusing (IEF), nonporous silica (NPS) reversed-phase high-performance liquid chromatography (RP-HPLC) and electrospray ionization time-of-flight mass spectrometry (ESI-TOFMS), respectively. Several hundred unique protein molecular weights were observed in a pI range from 4.8 to 8.5 and a mass range from 5 to 85 kDa. Proteins were positively identified by analysis of the pI (+/-0.5 pI units), an intact protein molecular weight (+/-150 ppm), and peptide mass mapping results. Using the molecular weight (MW) and peptide mapping results of identified proteins it was possible to characterize their posttranslational (PTMs) and/or sequence modifications. PTMs were detected on both forms of cytosolic actin, heat shock 90 beta, HINT and alpha-enolase. Sequence modifications or conflicts were observed for beta-and gamma-actin, ATP beta-synthase and heat shock 90 beta. IEF-NPS-RP-HPLC/ESI-TOFMS was used to determine experimental pI, MW and relative hydrophobicity values for each protein detected. This data was used to generate a 2-D pI-MS protein map, where proteins are displayed according to their pI and molecular weight. Protein molecular weight peaks are represented as bands in the 2-D pI-MS image where the gray scale of each band is proportional to the intensity of the protein molecular weight peak. In addition, a third hydrophobicity dimension (%B) was added as the % acetonitrile elution to generate a 3-D pI-MS-%B plot where each protein can be tagged according to three parameters.     

Quantification of carbonylated proteins in rat skeletal muscle mitochondria using capillary sieving electrophoresis with laser-induced fluorescence detection

Carbonyl-modified proteins are markers of oxidative damage. Here, we report a new method for detecting and quantifying carbonylated proteins by capillary sieving electrophoresis (CSE) with LIF detection (CSE-LIF). Alexa 488 hydrazide is used for the specific labeling of carbonyls while 3-(2-furoyl) quinoline-2-carboxaldehyde (FQ) is used for protein labeling. BSA subjected to metal-catalyzed oxidation is used to optimize the labeling reactions, confirm the separation power of CSE, and characterize the response of the LIF detector. The method is capable of detecting femtomole (fmol) amounts of carbonyls in proteins with molecular masses ranging from 26 to 30 kDa. Using this method, we determined that mitochondrial proteins isolated from skeletal muscle contains 2.1 +/- 0.1 (average +/- SD; n = 3) nmol carbonyl/mg protein. The methodology described here should be compatible with the analysis of single cells and needle biopsies taken from oxidative stress animal models.     

Copper fractionation by SEC-HPLC and ETAAS: study of breast milk and infant formulae whey used in lactation of full-term newborn infants

This method will allow the determination of bound copper to low relative molecular mass compounds in milk. The milk whey obtained by ultracentrifugation was submitted to fractionation by size exclusion chromatography (SEC) on a TSK-Gel2000 (Toso Haas) column with a mobile phase of 0.2 M NH4NO3 + NH3, pH 6.7. Fractions of effluent corresponding to the protein peaks were collected and the copper content was determined by ETAAS. The method was sensitive (LOD 0.4 microgram l-1 and LOQ 1.5 micrograms l-1 in the fraction; LOQ 7.5-22.5 micrograms l-1 referred to the milk sample and depended on fraction volume) and precise (RSD +/- 10%). Media sample recoveries from the column were 101.2%. Cu was predominantly present in fractions corresponding to relative molecular mass 76 and 15 kDa of breast milk while copper was mostly found in fractions corresponding to 14 and 38 kDa of cow's milk-based infant formulae; moreover, copper was eluted in the relative molecular mass region < 6 kDa.     

Theoretical and experimental investigation of the thermochemistry of CrO2(OH)2(g)

In this paper, we report the results of equilibrium pressure measurements designed to identify the volatile species in the Cr-O-H system and to resolve some of the discrepancies in existing experimental data. In addition, ab initio calculations were performed to lend confidence to a theoretical approach for predicting the thermochemistry of chromium-containing compounds. Equilibrium pressure data for CrO2(OH)2 were measured by the transpiration technique for the reaction 0.5Cr2O3(s) + 0.75O2(g) + H2O(g) = CrO2(OH)2(g) over a temperature range of 573 to 1173 K at 1 bar total pressure. Inductively coupled plasma atomic emission spectroscopy (ICP-AES) was used to analyze the condensate in order to quantify the concentration of Cr-containing volatile species. The resulting experimentally measured thermodynamic functions are compared to those computed using B3LYP density functional theory and the coupled-cluster singles and doubles method with a perturbative correction for connected triple substitutions [CCSD(T)].     

Preparative alkaline urea gradient PAGE: application to purification of extraordinarily-stable disulfide-linked homodimer of human growth hormone

The 40-60 pituitary human growth hormone (hGH) isoforms are so similar in their physico-chemical properties (charge, size, hydrophobicity) that the limited resolutions of chromatographic separation methodologies have not permitted most of them to be isolated. However, application of high-resolution preparative alkaline urea gradient PAGE has facilitated isolation of a disulfide-linked mercaptoethanol-resistant (MER) 45 kDa hGH dimer. Human pituitary extracts were separated by Sephadex G-100 chromatography under alkaline conditions. Pooled fractions containing MER-45 kDa hGH, as determined by SDS-PAGE, were then separated by Sephadex G-100 chromatography under acidic conditions followed by diethylaminoethyl (DEAE) anion-exchange chromatography. Pooled DEAE fractions containing MER-45 kDa hGH and other hGH isoforms were then separated by preparative electrophoresis in an alkaline polyacrylamide gradient (5-20%) slab gel containing 8 M urea into five distinct protein zones. One electroeluted zone contained pure MER-45 kDa hGH. The dimeric hGH isoform was immunoreactive at low concentrations (effective dose to produce 50% response (ED(50)) +/- S.E. = 58 +/- 5.00 pM) in a hGH radioimmunoassay, similar to that of standard monomeric hGH (ED(50) +/- S.E. = 22.93 +/- 3.90 pM), indicating that is was conformationally intact. Alkaline urea gradient PAGE is a valuable tool for preparative separation of structurally similar proteins such as isoforms of the hGH family.     

Electrophoretic studies of antimitochondrial antibodies: identification of mitochondrial antigens specific to primary biliary cirrhosis

Mitochondrial inner membrane proteins extracted from beef heart tissue were examined for reactivity to antimitochondrial antibody (AMA) present in sera of patients with primary biliary cirrhosis (PBC) by an immunoblotting technique. Four proteins, which reacted with AMA, had molecular masses of 70 kDa, 50 kDa, 47 kDa and 40 kDa, as defined by their relative mobility (Rf) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All sera of 114 PBC patients were positive with at least one and as many as four of the mitochondrial proteins. The major antigenic proteins of mitochondrial inner membrane to which AMA reacts were the 70 kDa and 47 kDa proteins. All PBC sera containing antibodies to the 50 kDa and/or 40 kDa proteins reacted with 70 kDa as well. The isolation of antigen reacting with AMA of PBC is important to warrant further study of AMA and the cause of the disease. The isolation of responsible antigens had been difficult because the four antigens were insoluble. However, the antigen newly found by us, the 36 kDa fragment, obtained by partial trypsin digestion, is soluble. Using several procedures, the antigenic protein target of AMA was purified from mitochondria for the first time. We determined the N-terminal sequence of the soluble 36 kDa fragment, 25 residues in length. Until now the N-terminal sequence of the 36 kDa protein has not shown significant homology with any known protein. The present results of antigen purification would contribute to the elucidation of the epitopes of AMA antigen.     

Validated LC–MS/MS method for simultaneous determination of doxorubicin and curcumin in polymeric micelles in subcellular compartments of MCF‐7/Adr cells by protein precipitation–ultrasonic breaking method

A specific and sensitive LC–MS/MS with protein precipitation– ultrasonic breaking method has been developed and validated for simultaneous determination of doxorubicin (DOX) and curcumin (Cur) in DOX and Cur co‐loaded hyaluronic acid–vitamin E succinatemicelles [(DOX + Cur)–polymeric micelles (PMs)] in subcellular compartments of resistant MCF‐7/Adr cells. Sequential extraction of four subcellular protein fractions (cytosolic, membrane/organelle, nucleic and cytoskeleton) was performed directly from MCF‐7/Adr cells after incubation with (DOX + Cur)–PMs. An ultrasonic breaking–methanol precipitation method was used for extraction of the fractions, and the micelle breaking efficiency with methanol was 98.1 and 97.6% for DOX and Cur, respectively. The analytes were analyzed using positive electrospray ionization coupled with multiple reaction monitoring. The calibration curves were linear over a concentration range of 0.5–400 ng/mL for DOX and 2–2000 ng/mL for Cur, and the recovery for the two analytes were >85% with negligible matrix effect. The intra‐day and inter‐day precision was <10.80% and relative error was within ±7.70%. The developed method was successfully applied for subcellular determination of DOX and Cur in MCF‐7/Adr cells. Moreover, Cur and (DOX + Cur)–PMs had a marked promoting effect on the distribution of DOX in the nucleic protein fraction.     

Assessment of metals bound to marine plankton proteins and to dissolved proteins in seawater

Studies based on laser ablation–inductively coupled plasma-mass spectrometry (LA–ICP-MS) have been performed to assess metal bound to dissolved proteins and proteins from marine plankton after two-dimensional polyacrylamide gel electrophoresis (2D PAGE). Dissolved proteins were pre-concentrated from surface seawater (60 L) by tangential ultrafiltration with 10 kDa molecular weight cut-off (MWCO) membranes and further centrifugal ultrafiltration (10 kDa) before proteins isolation by methanol/chloroform/water precipitation. Proteins isolation from plankton was assessed after different trichloroacetic acid (TCA)/acetone and methanol washing stages, and further proteins extraction with a phenol solution. LA–ICP-MS analysis of the electrophoretic profiles obtained for dissolved proteins shows the presence of Cd, Cr, Cu, and Zn in five spots analyzed. These proteins exhibit quite similar molecular weights (within the 10–14 kDa range) and pIs (from 5.8 to 7.3). Cd, Cr, Cu, and Zn have also been found to be associated to proteins isolated from plankton samples. In this case, Cd has been found to be bound to proteins of quite different molecular weight (9, 13 and 22 kDa) and pIs (4.5, 5.2, 5.5, and 10). However, trace elements such as Cr, Cu and Zn appear to be mainly bound to plankton proteins of low molecular weight and variable pI.     

Evaluation of the copper(II) complexing ability of ultrafiltered humic acid by the solvent extraction method.

A commercial humic acid dissolved in water was fractionated to nine samples by means of ultrafiltration (UF); the nominal molecular weight used for UF membranes was 1 k-200 kDa. Concerning the nine samples, copper(II) complexing capacities (CuCC) and conditional stability constants (beta) of the formed copper(II) complexes were measured by a solvent extraction method. A total organic carbon (TOC) and the UV-VIS absorption ratio (E350 nm/E450 nm) were also measured. From a comparison of these data, it was found that a) humic acids in each fraction formed two kinds of copper(II) complexes with different stability; b) the beta values obtained from each fraction were almost the same; c) large CuCC values were observed in the molecular weight range from 10 kDa to 20 kDa and below 1 kDa; d) molecules with molecular weight higher than 50 kDa scarcely had any copper(II) complexing ability; e) the values of CuCC/TOC of each fraction were in the range from 1.7 to 3.4 x 10(-7) mol mg(-1).     

Characterization of metabolic pathway of linoleic acid 9-hydroperoxide in cytosolic fraction of potato tubers and identification of reaction products

Potato tubers are shown to contain a unique lipoxygenase pathway to form 9-hydroperoxy-10,12-octadecadienoic acid (9-HPODE) from linoleic acid. Here, we report the metabolic pathway of 9-HPODE in the cytosolic fraction and the characterization of enzymes involved in the conversion of metabolites. The analysis of enzymatic reaction products at pH 5.5 revealed the formation of 9-keto-10,12-octadecadienoic acid, 9-hydroxy-10,12-octadecadienoic acid, 9,10-epoxy-11-hydroxy-12-octadecenoic acid, 9,10,13-trihydroxy-11-octadecenoic acid, and 9,12,13-trihydroxy-10-octadecenoic acid. The cytosolic enzymes were separated by anion-exchange chromatography into two fractions E1 and E2, having molecular masses of 66 and 54 kDa, respectively. The enzyme fraction E1 only produced 9-keto-10,12-octadecadienoic acid, whereas E2 formed other products. The enzyme E1 showed higher reactivity with 13- and 9-hydroperoxide of α-linolenic acid than 9-HPODE, but no reaction with hydroxy fatty acids. In contrast, the enzyme E2 showed the highest reactivity with 9-HPODE, followed by hydroperoxides of α-linolenic acid and arachidonic acid. We also evaluated the antibacterial activity of hydroxy fatty acids against Erwinia carotovora T-29, a bacterium infecting potato tubers. Growth of the bacteria was suppressed more potently with 9- or 13-hydroxy fatty acids than dihydroxy or trihydroxy fatty acids, suggesting a role for the metabolites in the resistance of bacterial infection.