Fractional Ethanol Precipitation Research Articles

Monosaccharide composition and bioactivity of tea flower polysaccharides obtained by ethanol fractional precipitation and stepwise precipitation

Yields of tea flower polysaccharides (TFPS) were investigated by ethanol fractional precipitation and stepwise precipitation and the monosaccharide composition of TFPS was investigated by ion chromatography(IC). Furthermore, a preliminary study on bioactivities of TFPS was discussed. The result indicated that the molecular weight distribution of TFPS was wide. Results of ethanol fractional precipitation and stepwise precipitation suggested that most of polysaccharides were precipitated when the final concentration of ethanol was adjusted to 30% (v/v). TFPS contained acid polysaccharides and it was composed of rhamnose, arabinose, galactose, glucose, xylose, mannose, GalA, and GluA with a molar ratio of 4.95:11.57:11.89:4.26:1.31:1.98:8.41:1.0. Furthermore, TFPS exhibited strong inhibitory effect on α-glucosidase and proliferation effect on lymphocytes.

Porphyran의 신속한 정제 방법 및 화학적 특성

A simple method for the purification of porphyran from laver Porphyra yezoensis was developed to obtain information for the development of food materials with biological functionality. Crude porphyran (CP) was extracted from dried laver in boiling water for 3 h, and then fractionated using cetylpyridinium chloride into an acidic fraction (CP-F1) and a neutral fraction (CP-F2). CP-F1 was fractionated further by fractional ethanol precipitation. Fraction CP-F1-70, precipitated at an ethanol concentration of 61-70% was the major fraction containing 68.1% of the yield from the initial fraction CP-F1. The CP-F1-70 fraction displayed a single band on Sepharose CL-4B with a molecular mass of 550 kDa, indicating a homogeneous polysaccharide. The molar ratio of galactose, 3,6-anhydro-L-galactose, 6-0-methyl-D-galactose and ester sulfate of CP-F1-70 was 1:0.32:0.07:0.53. This method is very useful for rapid and large-scale preparation of purified porphyran because it is compatible with mass production.

PURIFICATION AND PARTIAL CHARACTERIZATION OF A GLYCOPROTEIN ALPHA-AMYLASE INHIBITOR FROM WHITE KIDNEY BEAN (PHASEOLUS VULGARIS. L)

ABSTRACT A glycoprotein α-amylase inhibitor (α-AI) was extracted and purified from white kidney beans (Phaseolus vulgaris. L) by ethanol fractional precipitation, ion exchange chromatography and gel filtration column chromatography. The homogeneity of α-AI was demonstrated by gel filtration on sepharose CL-6B and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight was estimated to be about 36,000 Da by SDS-PAGE. The glycoprotein α-AI contained 88.2% protein and was rich in aspartic acid, glutamic acid, leucine, threonine and serine, with a trace amount of cysteine. The carbohydrate moiety, determined by reversed-phase high-performance liquid chromatography (RP-HPLC), consisted of Man, Glc, Gal and Xyl in a mole ratio of 2.42:1.50:1.52:1.00. The glycan and the core protein backbone was connected by O-linkage as determined by β-elimination reaction. PRACTICAL APPLICATION The α-amylase inhibitors are important because of their potential effects of insect control and crop plant defense against pests. The selective inhibition of human α-amylase also plays an important role in reducing digestive-starch degradation in patients suffering from diabetes and has high value in controlling human weight. In this paper, an α-amylase inhibitor that effectively inhibits porcine pancreatic and salivary amylases was isolated and purified from white kidney bean. Therefore, it may have high potential pharmaceutical value as a regulative agent in reducing digestive-starch degradation in patients suffering from diabetes and obesity.

A New Method of Purifying Fibrinogen with Both Biological and Immunological Activity from Human Plasma

Using a combination of Cohn ethanol fractionation, virus inactivation, glycine and sodium chloride precipitation, and lysine-Sepharose affinity chromatography, a unique and rapid simplified method was developed to obtain highly purified fibrinogen for diagnostic use with both biological (Clauss method) and immunological (Jacobsson method) activity. Yield was 0.66 g of fibrinogen per liter of starting pooled plasma, and the purified product showed good agreement in activity with the starting material. The purified fibrinogen solution contained over 95% clottable protein and had a clear appearance. No degradation was observed after urokinase treatment and the preparation provided good precision in fibrinogen measurement compared to pooled plasma. The simplified method was, thus, shown to result in a high-purity fibrinogen preparation, suitable for in vitro diagnostic use, as well as for use to prepare a fibrinogen reference material and to perform fibrinogen quality control using an automated coagulation analyzer.

Inhibitory activity of polysaccharide extracts from three kinds of edible fungi on proliferation of human hepatoma SMMC-7721 cell and mouse implanted S180 tumor.

AIM:To determine the activities of polysaccharide extracts from Flammulina velutipes (Curt. ex Fr.) Sing (FV), Lentinus edodes (LE)and Agaricus bispo-rus Sing (AB) on the proliferation of human hepatoma SMMC-7721 cells in vitro and on mouse implanted S-180 tumors in vivo.METHODS:The polysaccharide extracts were isolated from the fruit bodies of FV, LE and AB by the methods of hot-water extraction,Sevag's removal of proteins, ethanol precipitation, trypsin digestion and ethanol fractional precipitation. Human hepatoma SMMC-7721 cells were treated with 50mg/L polysaccharide extracts, and the mitosis index, mitochondria activity and cell proliferation were detected at different times in both control and experimental groups. The mice with S-180 implanted tumors were injected with the polysaccharide extracts at 24mg/kg body weight for 9d and the tumor weight was measured on the 15th day.RESULTS:The mitosis index of hepatoma cells in vitro could be significantly decreased by treatment with the polysaccharide extracts from the three kinds of edible fungi (P< 0.005). The cell numbers and mitochondria activity of SMMC-7721 cells treated with polysaccharide extracts were lower than those in control groups (P<0.005). The inhibition rates of polysaccharide extracts against implanted S-180 tumors in mice were 52.8%, 56.6% and 51.9% respectively compared with that in control groups.CONCLUSION:The polysaccharide extracts from the three kinds of edible fungi could inhibit not only the cultured malignant cells in vitro but also implanted S-180 tumor in vivo.

Inactivation and elimination of viruses during preparation of human intravenous immunoglobulin.

We report here the results of our evaluation of virus inactivation during the manufacturing steps of two intravenous immunoglobulin (IGIV) preparations. Virus inactivation and/or removal by processing steps, such as ethanol fractionation and polyethylene glycol precipitation, and deliberate virucidal steps, such as solvent/detergent treatment and pasteurization, were tested on a variety of human pathogenic and experimental model viruses, including human immunodeficiency, Hepatitis C, Mumps, Vaccinia, Chikungunya, Vesicular Stomatitis, Sindbis, and ECHO viruses. All viruses were successfully inactivated and/or eliminated by the processing steps studied. In some cases, however, multiple steps were required. We conclude that the incorporation of steps deliberately designed to inactivate or remove viruses during the production of IGIV provides an extra measure of viral safety.

Inactivation and Elimination of Viruses during Preparation of Human Intravenous Immunoglobulin

We report here the results of our evaluation of virus inactivation during the manufacturing steps of two intravenous immunoglobulin (IGIV) preparations. Virus inactivation and/or removal by processing steps, such as ethanol fractionation and polyethylene glycol precipitation, and deliberate virucidal steps, such as solvent/detergent treatment and pasteurization, were tested on a variety of human pathogenic and experimental model viruses, including human immunodeficiency, Hepatitis C, Mumps, Vaccinia, Chikungunya, Vesicular Stomatitis, Sindbis, and ECHO viruses. All viruses were successfully inactivated and/or eliminated by the processing steps studied. In some cases, however, multiple steps were required. We conclude that the incorporation of steps deliberately designed to inactivate or remove viruses during the production of IGIV provides an extra measure of viral safety.

Endogenous hepatic growth-modulating factors and effects of a choline-devoid diet and of phenobarbital on hepatocarcinogenesis in the rat.

The activities of an endogenous inhibitor and of a stimulator of cell proliferation were assayed in the livers of sham-operated (SO) or partially hepatectomized (PH) adult rats; rats fed a choline-supplemented (CS) or a choline-devoid (CD) diet; the same diets followed by acute CCl4 intoxication; the same diets supplemented with phenobarbital (PHB); or a CD diet containing DL-ethionine (ETH). The inhibitor and the stimulator were semipurified by fractional ethanol precipitation of a liver cytosolic fraction, and their activities were assessed by means of bioassays in vitro. The livers of SO rats and of rats fed the CS diet contained only inhibitor activity. Following PH, a CD diet, or CCl4 intoxication the inhibitor activity was suppressed, and there was a simultaneous appearance of a stimulator activity. Thus, PH, a CD diet, and CCl4 intoxication cause similar cellular (loss and regeneration) and humoral-homeostatic changes in adult rat livers. We propose that these changes constitute a basic attribute of the mechanism whereby the three conditions affect similarly hepatocarcinogenesis in the rat, especially in the case of a CD diet, because the changes it induces are chronic rather than acute. PHB, another promoter of chemical hepatocarcinogenesis, affected neither the inhibitor nor the stimulator activity. Thus, PHB seems to be acting by a different mechanism than that of the other three agents. ETH did not modify the shift in the balance of the growth-modulating factors induced by a plain CD diet. This shift may account for the marked stimulation of carcinogen-induced oval cell proliferation exerted by a CD diet. The significance of these results is discussed in the context of known effects of a CD diet and of PHB on hepatocarcinogenesis in rats.

Purification and characterization of the serotype c antigen from Actinobacillus actinomycetemcomitans.

The serotype c antigen from Actinobacillus actinomycetemcomitans was purified with fractional ethanol precipitation of cell-free culture supernatant, sequential ion-exchange chromatography, and gel filtration chromatography. The preparation obtained demonstrated a single precipitin line in immunodiffusion, immunoelectrophoresis, and crossed immunoelectrophoresis when rabbit antisera to serotype c whole bacterial cells were used. No immunological reaction was detected with antisera to serotype c lipopolysaccharide, indicating that lipopolysaccharide was not present in the preparation. The serotype c antigen was composed of 95% carbohydrate, 2% protein, and 3.1% phosphate. Gas chromatographic analysis of the antigen obtained from growth in either complex or chemically defined media revealed that the carbohydrate constituent was composed of 84 to 90.1% mannose, 4.8 to 16% glucose, 1.9% N-acetylglucosamine, 1.4% fucose, and 0.2% galactose. The present data suggest that A. actinomycetemcomitans serotype c antigen is predominantly a mannose-containing carbohydrate suggestive of a mannan.

Protective action of double-stranded interferon-inducing complexes against experimental encephalomyocarditis in mice

A study was made of the action of human gastric mucosa G1 and G2 chalones on cellular regeneration of mouse gastric mucosa and of the duration of their maximal effect. Chalone fractions were obtained from the mucous membranes of 21 stomachs resected for peptic ulcer by the method of fractional ethanol precipitation. The data indicate that the maximal inhibitory action of G1 chalone occurs in 3, whereas that of chalone G2 in 6 hours. Some specificity of the action of chalones was discovered depending on the part of the gastric mucosa from which they were obtained.

Isolation and partial characterization of an extracellular glucansucrase from Leuconostoc mesenteroides NRRL B-1355 that synthesizes an alternating (1→6), (1→3)-α- d-glucan

Leuconostoc mesenteroides NRRL B-1355 grows on sucrose to produce two extracellular α- d-glucans. Although both are termed dextrans, they are chemically and physically distinct, and can be separated by fractional ethanol precipitation into fractions designated L and S. Fraction L is similar to B-512F dextran, having 95% α-(1→6) linkages and 5% α-(1→3) branch linkages, but fraction S has an alternating sequence of α-(1→6) and α-(1→3) linkages. Because of its structural differences from dextran, its different physical characteristics, and its resistance to hydrolysis by endodextranase, we have named glucan S, alternan, and the enzyme that synthesizes it from sucrose, alternansucrase. Alternansucrase has been isolated by two different methods. The first involves removal of the fraction L glucan from the culture fluid via hydrolysis by an endodextranase, followed by chromatography on Bio-Gel A5m. The void-volume fraction synthesizes only alternan, whereas the slower-migrating, second fraction synthesizes mainly dextran, together with some alternan. The second method utilized hydrophobic chromatography on O-(phenoxyacetyl)cellulose; a portion of the alternansucrase did not bind, whereas the bound portion, removed by eluting with detergent, contained both alternansucrase and dextransucrase. The glucans were identified by physical appearance, the concentration of ethanol required for precipitation, periodate-oxidation behavior, and susceptibility to hydrolysis by endodextranase. Also studied was the inhibition of the enzymes by 3-deoxy-3-fluoro-α- d-glucopyranosyl fluoride, tris(hydroxymethyl)aminomethane, 2-aminoethanol, and octyl β- d-glucopyranoside.

Purification and properties of brain alkaline phosphatase.

Abstract— Alkaline phosphatase from sheep brain has been purified to homogeneity. The method includes butanol extraction, fractional ethanol precipitation, ion‐exchange chromatography on DEAE‐cellulose, and on DEAE‐Sephadex followed by Sephadex G‐200 filtration. By these steps, the enzyme is purified 22,920‐fold with 15% recovery. The homogeneous enzyme is shown to be a sialoglycoprotein in nature. Neuraminidase treatment reduces the electrophoretic mobility of the enzyme. The enzyme shows pyridoxal phosphate phosphatase activity along with p‐nitrophenylphosphate phosphatase activity. Both these compounds behave as mutual alternate competitive substrates. The general properties of the enzyme are described.

Separation of Two Phosphorylase Kinase Phosphatases from Rabbit Skeletal Muscle

Cyclic-AMP-dependent protein kinase catalyses the activation of phosphorylase kinase and the phosphorylation of two serine residues on the alpha subunit and beta subunit of phosphorylase kinase [Cohen, P., Watson, D.C. and Dixon, G.H. (1975)]. The dephosphorylation of phosphorylase kinase has been shown to be catalysed by two distinct enzymes, termed alpha-phosphorylase kinase phosphatase and beta-phosphorylase kinase phosphatase. These two enzymes show essentially absolute specificity towards the alpha and beta subunits respectively. The two phosphatases copurified through ethanol fractionation, DEAE-cellulose chromatography and ammonium sulphate precipitation, but were separated from each other by a gel filtration on Sephadex G-200. alpha-Phosphorylase kinase phosphatase was purified 500-fold from the ethanol precipitation step, and beta-phosphorylase kinase phosphatase 320-fold. The molecular weights estimated by gel filtration were 170--180 000 for alpha-phosphorylase kinase phosphatase and 75--80 000 for beta-phosphorylase kinase phosphatase. Since the activity of phosphorylase kinase correlates with the state of phosphorylation of the beta subunit (Cohen, P. (1974)), beta-phosphorylase kinase phosphatase is the enzyme which reverses the activation of phosphorylase kinase. alpha-Phosphorylase kinase phosphatase is an enzyme activity that has not been recognised previously. Since the role of the alpha-subunit phosphorylation is to stimulate the rate of dephosphorylation of the beta subunit (Cohen, P. (1974)), alpha-phosphorylase kinase phosphatase can be regarded as the enzyme which inhibits the reversal of the activation of phosphorylase kinase. The implications of these findings for the hormonal control of phosphorylase kinase activity by multisite phosphorylation are discussed.

Immunochemistry of purified polysaccharide type antigens of group B streptococcal types Ia, Ib, and Ic.

The HCl-extracted purified polysaccharide type antigens of group B Streptococcus types Ia and Ib were composed of galactose and N-acetylglucosamine in a molar ratio of 3:1 for the Ia antigen and 2:1 for the Ib antigen. Immunochemical data were the same for the Ia antigens of type Ia, purified in this study, and type Ic, purified earlier. Glucosamine inhibited the Ib quantitative precipitin reactions more effectively than did N-acetylglucosamine, whereas the reverse was true of the Ia reaction. Ouchterlony studies were consistent with these observations and also revealed two type-specific precipitin bands with the HCl-extracted Ia antigens. All saline-extracted type antigens, however, formed single Ouchterlony bands that were only partially identical to the corresponding HCl antigens. Purification of the saline antigens was accomplished by treatment with cold trichloroacetic acid and by fractional ethanol precipitation. Immunoelectrophoresis experiments showed that the saline antigens were more negatively charged than the HCl antigens. The presence of sialic acid in the saline antigens probably accounted for their net negative charge and the fact that they were partially degraded by mild acid hydrolysis. The same serological specificities were observed with saline- and with HCl-extracted antigens.

Purification and properties of an inhibitor of the tryptophan-synthase-inactivating enzymes in yeast.

An inhibitor of the tryptophan‐synthase‐inactivating enzymes has been isolated from bakers' yeast and purified 27‐fold on a protein basis, by heat treatment, precipitation by trichloroacetic acid, ethanol fractionation and acid precipitation. The inhibitory activity of the purified material is not affected by incubation with RNAase, DNAase, or muramidase, but is destroyed by incubation with pronase or trypsin. Inhibitory activity depends, therefore, on the protein content of the material.The extent to which the inactivating enzymes are inhibited is proportional to inhibitor concentration. Tryptophan‐synthase inactivases I and II are both inhibited. The inhibitor appears to be specific in its action: it is much more effective than soybean trypsin inhibitor in inhibiting the inactivases, but has comparatively little effect on the esterolytic activity of trypsin.Inhibitory activity was measured in crude extracts of Saccharomyces cerevisiae growing in batch culture. In the exponential growth phase, the inhibitor was barely detectable, but its levels subsequently rose at the end of the exponential phase and in the stationary phase. The possible biological significance of these observations is discussed.

The fate of gonococci in polymorphonuclear leucocytes.

SUMMARY The fate of three freshly isolated strains of N. gonorrhoeae following phagocytosis by human or guinea-pig polymorphs was studied in vitro. Less than 1 per cent. of gonococci phagocytosed by the guinea-pig cells survived at 100 min., but some 20 per cent. of gonococci associated with the human polymorphs were not killed even after 3 hr. The persisting gonococci were destroyed when the polymorphs were exposed to penicillin and this indicates that the surviving bacteria lay outside the polymorph membrane. Cationic proteins prepared by fractional ethanol precipitation of acid extracts of the granules from human and guinea-pig polymorphs rapidly destroyed gonococci in vitro and, presumably, are partly responsible for the ability of polymorphs to kill gonococci. Although these findings require confirmation in studies with gonococci grown in vivo, they strongly suggest that man’s susceptibility to gonorrhoea is not explained simply by the survival of gonococci in polymorphs.

Somatic antigens of Streptococcus group E. II. Separation and a partial physicochemical characterization.

Antigenic extracts of Streptococcus group E (SGE) were subjected to fractional ethanol precipitation, block (preparative) electrophoresis, and gel filtration for the purpose of separating the type antigen from the group antigen. Ethanol precipitation was ineffective in separating the substances. Block electrophoresis yielded serologically pure group antigen and a mixture of type and group antigen. Serologically pure type antigen was obtained by gel filtration. In some cases, pure group antigen was also recovered; in others, it was contaminated with type antigen. Gel filtration column effluents of antigenic extracts of SGE serotypes, I, II, III, IV, V and "untypable" isolates, collected from the region in which type antigen was eluted, were studied by paper chromatography and infrared spectrophotometry in an effort to develop a nonserological means of detecting type antigen. Hydrolysates of type antigens or suspect type antigens of serotypes I through V contained l-rhamnose, d-glucose, and several unidentified substances. d-Galactose also was present in hydrolysates of serotypes III and V. Untypable isolates and negative controls contained traces of d-glucose only. The data suggested that serotypes I through V contained a type antigen and that the untypable isolates were devoid of type antigen. Infrared absorbance spectra seemed to support the paper chromatography data.

Immunochemical studies on blood groups. XVIII. Fractionation of hog gastric mucin and individual hog stomach linings

1. 1. Blood-group substances were prepared from pooled hog gastric mucin, and from individual hog stomach linings after autolysis and peptic digestion, by the phenol extraction and fractional ethanol precipitation method of Aminoff, Morgan, and Watkins. The analytical and immunochemical properties of these fractions were compared with the properties of substances purified by the earlier phenol-ethanol method of Morgan and King. No significant separation of the A and O(H) substances in pooled hog gastric mucin was achieved by the fractional ethanol precipitation procedure. The use of either method to prepare blood-group A substance from single A hog stomachs devoid of O(H) activity resulted in products which contained similar amounts of blood-group A antigen, as judged by quantitative immunochemical methods. In the fractionation of single hog stomach linings, whether A or O(H), the dispersion of active material in the phenol-ethanol system appeared to depend on the completeness of peptic digestion. 2. 2. An acid-soluble and an acid-insoluble fraction prepared from hog gastric mucin by ethanol precipitation and electrodecantation by Holzman and Niemann differed in no important respect from fractions of gastric mucin purified by either phenol-ethanol procedure.