Soluble High Molecular Weight Fractions Research Articles

Effect of cadmium binding form on rabbit aminolevulinate dehydratase in high molecular weight fraction of rainbow trout liver

Enzyme inhibition owing to cadmium (Cd) accumulation in the soluble high molecular weight fraction (HMF) of the liver from rainbow trout was investigated by measuring the inhibition of aminolevulinate dehydratase (ALAD). Factors affecting ALAD, which reacts specifically to metal, were examined before measuring ALAD activity. Although enzyme inhibition by Cd in the HMF was confirmed in the excretion 2 (E2) group fed Chinese parsley (CP), it was weaker than that in the excretion 1 (E1, control) group, probably due to the chelation of Cd by some substances contained in CP. Based on the absorption spectrum, the proportion of Cd bound to cysteine thiol radicals in the HMF of the E2 group was greater than that of E1. The HMF might detoxify harmful metals to some extent because it contains relatively higher levels of cysteine residues than metallothionein (MT) and shows comparatively weak enzyme inhibition. The results of the present study demonstrated that, in addition to MT, HMF is involved in the detoxification of harmful heavy metals in fish.

Detection of d-amino acids in purified proteins synthesized in Escherichia coli

It has long been believed that amino acids comprising proteins of all living organisms are only of the L-configuration, except for Gly. However, peptidyl D-amino acids were observed in hydrolysates of soluble high molecular weight fractions extracted from cells or tissues of various organisms. This strongly suggests that significant amounts of D-amino acids are naturally present in usual proteins. Thus we analyzed the D-amino acid contents of His-tag-purified beta-galactosidase and human urocortin, which were synthesized by Escherichia coli grown in controlled synthetic media. After acidic hydrolysis for various times at 110 degrees C, samples were derivatized with 4-fluoro-7-nitro-2, 1, 3-benzoxadiazole (NBD-F) and separated on a reverse-phase column followed by a chiral column into D- and L-enantiomers. The contents of D-enantiomers of Ala, Leu, Phe, Val, Asp, and Glu were determined by plotting index D/(D + L) against the incubation time for hydrolysis and extrapolating the linear regression line to 0 h to eliminate the effect of racemization of amino acids during the incubation. Significant contents of D-amino acids were reproducibly detected, the D-amino acid profile being specific to an individual protein. This finding indicated the likelihood that D-amino acids are in fact present in the purified proteins. On the other hand, the D-amino acid contents of proteins were hardly influenced by the addition of D- or L-amino acids to the cultivation medium, whereas intracellular free D-amino acids sensitively varied according to the extracellular conditions. The origin of these D-amino acids detected in proteins was discussed.

Effect of coffee Melanoidin on human hepatoma HepG2 cells. Protection against oxidative stress induced by tert‐butylhydroperoxide

Soluble high-molecular weight fraction (named melanoidin) from coffee brew was isolated by ultrafiltration, subsequently digested by simulating a gastric plus pancreatic digestive condition and partly characterized by CZE, gel-filtration and browning. The objective of the present study was to investigate the potential protective effect of the coffee melanoidin submitted to gastrointestinal digestion on cell viability (lactate dehydrogenase leakage) and redox status of cultured human hepatoma HepG2 cells submitted to oxidative stress induced by tert-butylhydroperoxide (t-BOOH). Concentration of reduced glutathione (GSH) and malondialdehyde (MDA), generation of reactive oxygen species (ROS) and activity of antioxidant enzymes glutathione peroxidase (GPx) and reductase (GR) were used as markers of cellular oxidative status. Pretreatment of cultured HepG2 cells with 0.5-10 microg/mL digested coffee melanoidin (DCM) for 2 or 20 h completely prevented the increase in cell damage and GR and partly prevented the decrease of GSH and the increase of MDA and GPx evoked by t-BOOH in HepG2 cells. In contrast, increased ROS generation induced by t-BOOH was not prevented when cells were pretreated with DCM. The results show that treatment of HepG2 cells with concentrations of DCM within the expected physiological range confers the cells a significant protection against an oxidative insult.

Assessing the non-specific hydroxyl radical scavenging properties of melanoidins in a Fenton-type reaction system

Soluble high molecular weight fraction (>10 kDa, named melanoidins) was isolated from Maillard reaction model systems, coffee, beer and sweet wine by ultrafiltration. Deoxyribose method was adjusted for measuring the hydroxyl radical scavenging properties of melanoidins. The presence of competitive melanoidins with deoxyribose for OH decrease the rate of deoxyribose degradation. Possible interferences to the deoxyribose method have been evaluated. Most of isolated melanoidins exhibited a variable and measurable non-site-specific hydroxyl scavenging activity in a Fenton-type reaction system. The iron reducing properties of melanoidins at the reaction conditions were evaluated with ferrozine. It has established a kinetic approach to assess the second rate constants of hydroxyl radical scavenging reactions of melanoidins. This approach may be a valuable tool for addressing the structure–activity relationships of melanoidins in a future. There is no correlation between browning (absorbance at 420 nm) and efficiency for scavenging hydroxyl radicals in solution.

Iron-binding ability of melanoidins from food and model systems

Soluble high molecular weight fractions isolated from the development of the Maillard reaction in 22 different model systems and food matrices were studied in order to assess their iron-chelating ability in vitro. Melanoidins and iron were incubated in a Na-acetate buffer (0.1 M, pH 5) at different weight ratios and free iron was measured by FAAS and the bathophenantroline procedure at the steady state. Melanoidins were classified at three levels according to their number of co-ordination sites for iron. Melanoidins from coffee (medium roasted), different types of beer (Pilsener, Abbeys, and dry-stout styles), and sweet wine (Pedro Ximenez) exerted a low iron-binding effect compared with melanoidins from model systems. The type of sugar was shown to be a significant parameter for obtaining melanoidins with high iron ability, and glucose was more efficient than lactose. No relationship was observed between browning and iron binding ability of melanoidins from model systems. The chromophoric residues were not the main co-ordination sites for iron complexation in the melanoidin structure.

Genotoxicity and mutagenicity of melanoidins isolated from a roasted glucose–glycine model in human lymphocyte cultures, intestinal Caco-2 cells and in the Salmonella typhimurium strains TA98 and TA102 applying the AMES test

Melanoidins are formed during household cooking procedures and are part of our daily diet, but data on their toxicological potential are still scarce. Therefore, the mutagenic, cytotoxic and genotoxic activity of the water soluble total fraction (sol A), the water soluble high molecular weight fraction (HMW; Molecular weight > 12,400 Da) and the remaining water soluble low molecular weight fraction (LMW) isolated from a glucose–glycine model system roasted at 125 °C was comprehensively studied in human lymphocytes (genetic end point: sister chromatid exchange (SCE)), Caco-2 cells (SCE, cell viability, cell proliferation) and in the Salmonella typhimurium strains TA98 and TA102 (Ames test). Tests were performed in a dose- and time-dependent manner. The results indicate a significant increase in SCE formation in human lymphocytes after the exposure to 0.05% and 0.1% of the melanoidin fractions. In Caco-2 cells, only the exposure to LMW increased the SCE formation as a matter of concentration. Cell's proliferation and viability decreased significantly after exposure to melanoidins. In the AMES test, melanoidins did not show a mutagenic potential, neither using the TA98 nor the TA102 strain. These results show that melanoidins isolated from the glucose–glycine mixture exhibited modest but significant genotoxic effects in human lymphocytes and, in particular the LMW, in Caco-2 cells, but they induce neither in low nor in very high concentrations mutagenicity in bacteria strains.

Peroxyl radical scavenging activity of soluble high molecular weight fraction from coffee brews

Roasting process of coffee beans affects the final composition of coffee brew. While the presence of some of its antioxidant compounds (phenolic acids) decrease throughout the treatment, melanoidins are formed during the thermal process. Recent research into nutritional, physiological and functional properties of melanoidins has suggested that they have antioxidant activity. Instant coffees produced from roasted coffee beans were obtained from a company coffee in three different roasting degrees: light (CTn 110), medium (CTn 85) and dark (CTn 60). Melanoidins of high molecular weight (> 10 000 Da) were obtained from each coffee, as well as pure melanoidins by incubation in 2M ClNa and ultrafiltration. CGA contained in extractes of different melanoidins (1 mg/ml) was measured by HPLC. Peroxyl radical scavenging activity of melanoidins and pure melanoidins were tested using a procedure to inhibite the peroxidation of linoleic acid. Antioxidant efficiency (AE<sub>50</sub>, ml/min/mg) was calculated as the slope of the time of inhibition at 50% (min) against concentration (mg/ml). No significant differences were found between melanoidins and pure melanoidins in the different roasting degrees. Peroxyl radical scavenging activity of melanoidins from coffee brew is mainly due to the chelated compounds by ionic binds.

Characterization of a water-soluble polysaccharide fraction with immunopotentiating activity from Bifidobacterium adolescentis M101-4.

The soluble and insoluble fractions obtained after sonication and centrifugation of Bifidobacterium adolescentis M101-4 cells were examined, and both of these fractions exhibited mitogenic activity in an assay of murine splenocytes and Peyer's patch cells in vitro. The soluble fraction was further treated by a 6-step procedure involving proteinase K-treatment, ultrafiltration with a 50-kDa cut-off molecular-sieving membrane, anion-exchange chromatography, dialysis, ultrafiltration through a 6-kDa cut-off membrane filter, and gel-filtration to yield a soluble high molecular weight fraction (SHF) which was effective for stimulating the proliferation of murine splenocytes. Almost three quarters of this fraction by weight was found to consist of carbohydrates containing glucose and galactose as major constituents, and the average molecular weight was estimated to be between 60,000 and 2,460,000, with the main peak at 1,550,000 Da, by the retention time of gel permeation chromatography. A structural analysis by 1H- and 13C-nuclear magnetic resonance and methylation indicated that SHF contained polysaccharides consisting of -4Galp1-, -4Glcp1-, and -6Glcp1- as the major residues, and Galf1- and -6Galf1- as the minor residues. Immunopotentiating SHF was found to contain galactofuranosyl residues as characteristic constituents which had not been previously detected in other soluble fractions from Gram-positive bacteria.

Non-allosteric regulation of the uridine kinase from seeds of Zea mays

Uridine kinase (ATP: uridine 5′-phosphotransferase, EC 2.7.1.48) has been partially purified from ungerminated hybrid corn seed. It is associated with a soluble high molecular weight fraction from which it apparently cannot be dissociated without loss of activity. The stability of the enzyme is enhanced by the addition of dithiothreitol, glycerol and nucleotide substrate. The nucleoside specificity of the enzyme is limited to nucleosides containing pyrimidine and ribose moieties, such as uridine and cytidine. High concentrations of nucleosides cause substrate inhibition, however. The K m values for uridine and cytidine are 53 μM and 125 μM, respectively, and under subsaturating conditions uridine is phosphorylated about five times faster than cytidine. The reaction follows an ordered Bi Bi Kinetic pattern, with ATP and ADP in competition for the free form of the enzyme. Purine, but not pyrimidine, nucleoside triphosphates serve as phosphate donors without regard to the sugar moiety. However, all of these triphosphates appear to compete for the same site on the enzyme. ( K m ATP = 590 μM, K m (app) GTP = 61 μM , and CTP and UTP are linear competitive inhibitors against ATP, with K i values of 60μM and 240 μM, respectively.) Therefore, end product control of uridine kinase apparently does not involve allosteric sites, but instead is envisioned as simple competition between relatively effective or ineffective phosphate donors for a position on the enzyme.