The antigenic structure ofFrancisella tularensis

The ether antigen ofFrancisella tularensis was fractionated using DEAE-cellulose and CM-cellulose chromatography, and Sephadex G-200 gel filtration. The CM-cellulose chromatography did not appear to be a suitable method for separation of individual components of the complex ether antigen. Most of the polysaccharide material and substances with a high phosphorus content were eluted already in the first peak of the elution curve. This method could be used only to separate a component, yielding one immunoelectrophoretic precipitin line, localized towards the cathode. On the other hand, the DEAE-cellulose chromatography, and particularly the Sephadex G-200 gel filtration (especially when recycling was introduced), yielded a clear separation of relatively clean components of the ether antigen. The present work provides a comparison between the immunochemical (immunoelectrophoretic) properties of fractions obtained by these methods and components isolated by salting out with ammonium sulphate, ethanol or trichloracetic acid precipitation.     

Enhanced antibody response to a detergent-soluble antigen in human filariasis after treatment with diethylcarbamazine

An antigen fraction has been isolated from the water insoluble component of cattle filarial parasiteSetaria digitata by detergent NP-40 solubilization, precipitation with ammonium sulphate and fractionation on sephadex G-100. Immunoglobulin G response to the isolated antigenic fraction was selectively suppressed in asymptomatic microfilaraemic people in comparison to the amicrofilaraemic groups of endemic normals and chronic patients. However, treating microfilaraemic people with diethylcarbamazine enhanced the antibody levels by 10-fold. These results suggest that active infection suppresses the response induced by the isolated antigenic fraction which is elevated after clearance of microfilariae.     

Staphylococcal β-Hemolysin I. Purification of β-Hemolysin

The purification of staphylococcal β-hemolysin was accomplished by the successive use of three protein fractionation methods. The first method employed was a double precipitation with the use of ammonium sulfate at 65% saturation. The second phase of purification used Sephadex G-100 column fractionation. The third phase utilized either carboxymethyl cellulose or diethylaminoethyl cellulose fractionation. The last two fractionation methods both resulted in the separation of a relatively high concentration of cationic hot-cold lysin and a low concentration of anionic hot-cold lysin. Because of the low concentration of the anionic component, its purity could not be assessed. However, the purity of the cationic component was demonstrated by immunodiffusion, microimmunoelectrophoresis, and by disc polyacrylamide gel electrophoresis. In addition, antisera against purified cationic β-hemolysin yielded one line of precipitate when tested against the original crude β-hemolysin. The purified cationic β-hemolysin was stable in the lyophilized state. Crude β-hemolysin was dermonecrotic, whereas purified cationic β-hemolysin was not dermonecrotic even after Mg⁺⁺ activation.     

THE IMMUNOLOGICAL SPECIFICITY OF THE EUGLOBULIN AND PSEUDOGLOBULIN FRACTIONS OF HORSE AND H^MAN SERUM

That portion of horse and human serum globulin precipitated by 33 per cent saturation with ammonium sulfate and precipitated on subsequent dialysis was taken as euglobulin; and the fraction precipitated between 33 and 50 per cent saturation and remaining in solution on subsequent dialysis was taken as pseudoglobulin. The sera of rabbits injected with either of these antigens gave precipitation with both. However, two distinct and fraction-specific antibodies could be demonstrated by absorbing the sera with the one antigen, and testing the supernatant fluid with the other. The experimental results are adequately explained on the basis that there are at least two antigenically distinct globulins in serum which we may term globulin I and globulin II and which are largely associated with so called euglobulin and pseudoglobulin respectively. The ordinary methods of salting out and dialysis do not effect complete separation and each globulin preparation contains a trace of the other antigen. The antisera to these euglobulin and pseudoglobulin preparations therefore contain antibodies to both antigens. Each protein solution precipitates all the antibody specific for the one antigen and in addition, by virtue of the trace of contaminating protein, precipitates a portion, and only a portion of the antibody specific for the other antigen. The fact that antisera to whole serum contain these same fraction-specific antibodies suggests that this immunological specificity is an inherent property of two globulins present as such in serum and is not an artifact induced by their precipitation and purification. Lipoids extracted from the globulins by ether, petroleum ether, and alcohol give no demonstrable reaction with antisera to these globulins; antisera absorbed with a large excess of lipoid are not affected as regards their reactivity with the original protein; and globulins extracted with ether and petroleum ether at room temperature are not affected as regards their reactivity with antisera. It is concluded that the immunological specificity of the globulin fractions as evidenced by the precipitation reaction is not determined by lipoids associated with the protein.     

Purification and properties of arylsulphatase from the brain of the silkworm Bombyx mori

(1) Arylsulphatase of the silkworm Bombyx mori was partially purified using ammonium sulphate fractionation, ethanol precipitation, Sephadex G-200 gel filtration and Con-A Sepharose chromatography. (2) The purified enzyme preparation was not homogeneous but showed no beta-glucuronidase or beta-galactosidase activities. (3) The kinetic properties of the enzyme indicated that it could be classified under type-2 arylsulphatases of vertebrates. (4) The purified enzyme shows very little activity towards p-nitrophenyl sulphate and none towards cerebroside 3-sulphate.     

Preparation and properties of fibrinolytic enzymes produced by Cochliobolus lunatus

Some properties of the crude lyophilized fibrinolytic enzyme produced by Cochliobolus lunatus in surface culture were studied. Enzyme concentrations over the range from 0.16 to 10.16 mg/mL showed that concentration above a certain level ceased to be the limiting factor controlling enzyme action. At pH 6.8 and a temperature of 40°C, the fibrinolytic enzyme showed maximal activity at a human fibrin concentration of 2 mg/mL. The optimum pH values for enzyme activity were 6.98 and 7.0, using Sørensen and Mcllvaine buffers, respectively. Fibrinolytic enzymes were isolated from a static culture of Cochliobolus lunatus; isolation was carried out with various agents. Ammonium sulphate yielded the highest recovered fibrinolytic activity. The fraction salted out by precipitation at 25% ammonium sulphate saturation possessed the highest recovered fibrinolytic activity compared to the ammonium sulphate, ethanol, and acetone fractions.     

Lipase from Pseudomonas fragi. I. Purification of the Enzyme

The experimental conditions required to isolate a lipase from Pseudomonas fragi were determined. The organism was grown in a buffered tryptone medium for 4 to 5 days at 20 C. The lipase in the culture supernatant fluid was isolated by fractionation with ammonium sulfate at 60% saturation, followed by acetone precipitation at 30-60% concentration. Further purification was made by using Sephadex G-200 gel-filtration and diethylaminoethyl cellulose chromatography. Electrophoretic analysis of the purified lipolytic fraction showed apparent homogeneity by both cellulose polyacetate and disc electrophoresis. The specific activity of the purified enzyme was about 100 times that of the starting culture filtrate, and the yield was about 1.8% of the original activity.     

Staphylococcal Hyaluronate Lyase: Purification and Characterization Studies

Staphylococcal hyaluronate lyase (hyaluronidase) derived from a pathogenic strain of staphylococcus was purified by means of salt fractionation with ammonium sulfate and gel filtration through Sephadex G-100. Most of the enzyme activity from concentrated culture supernatant fluids of staphylococci was obtained in a fraction precipitated by 90 to 100% saturation with ammonium sulfate. A small amount of enzyme was also precipitated by 80 to 90% saturation with the salt. The hyaluronidase-rich fractions did not contain other staphylococcal enzymes, such as coagulase, protease, lipase, and staphylokinase. These enzymes were present in the original concentrates. Molecular sieving chromatography of the partially purified enzyme by filtration through Sephadex G-100 resulted in a further increase in specific enzyme activity. However, more than one active peak was obtained after gel filtration, thus suggesting that there may be more than one molecular form of the enzyme. Immunodiffusion in agar gel of the chromatographically purified enzyme fraction, with immune serum from rabbits injected with concentrated staphylococcal culture supernatant fluids, indicated that there was one major antigen. A similar antigen, giving reactions of identity with the purified material, was present in the original culture supernatant fluid.     

Cellulolytic enzyme system of Trichoderma koningii. Separation of components attacking native cotton

1. Cell-free culture filtrates from Trichoderma koningii were concentrated by precipitation with ammonium sulphate between the limits of 20% and 80% saturation. 2. Removal of a low-molecular-weight carboxymethylcellulase (CM-cellulase) component by chromatography on Sephadex G-75 had no effect on the ability of the enzyme complex to solubilize cotton. 3. Further chromatography on DEAE-Sephadex separated a component (C(1)) from the C(x) (CM-cellulase) and beta-glucosidase activities. Separately these components had little ability to produce soluble sugars from cotton, but when recombined in their original proportions this capacity was almost completely recovered. 4. The C(x) component was further fractionated on SE-Sephadex into a fraction containing only CM-cellulase and a fraction showing CM-cellulase and beta-glucosidase activities: the latter two components could be separated by heat treatment. 5. The C(1) component had no swelling factor (S-factor) activity (Marsh, Merola & Simpson, 1953; Reese & Gilligan, 1954) on its own, but it had a synergistic effect on the S-factor activity associated with the CM-cellulase and beta-glucosidase components.     

Echinococcus granulosus: comparison between antigens in scolices and hydatid fluid.

The sheep hydatid fluid and scolex antigens of Echinococcus granulosus were precipitated by increasing ammonium sulphate concentrations. The antigenic profiles, obtained by complement fixation and indirect hemagglutination inhibition tests on the ammonium sulphate precipitates after linear sucrose gradient ultracentrifugation, were different comparing the hydatid fluid and the scolex extracts. Antigenic non-identity was found between sheep hydatid fluid and scolex extracts by immunodiffusion and indirect hemagglutination inhibition tests. The ammonium sulphate precipitates of hydatid fluid and scolex extracts revealed several different bands by slab-gel examination.     

Isolation of fractions from Bordetella parapertussis extracts and their use for preparing an erythrocyte diagnostic preparation]

Soluble parapertusis antigen, serologically active, having hemosensitive properties and containing only 2 antigenic components was obtained by the method of ethanol fractionation of microbial extracts. This method is simple and convenient for production purposes. The antigen thus obtained was used for the production of a highly specific erythrocytic diagnostic preparation (formalinized, liquid). When tested in reaction with animal and human sera, the new diagnostic preparation proved to be sufficiently active and species specific.     

东亚飞蝗谷胱甘肽S-转移酶分离纯化

通过硫酸铵沉淀技术和GSH-agarose亲和层析对东亚飞蝗Locusta migratoria manilensis(Meyen)5龄若虫谷胱甘肽S-转移酶(glutathione S-transferases,GSTs)进行了分离纯化。结果表明GSTs活性在硫酸铵各沉淀段均有分布,但在55%～100%沉淀段活性较高,在硫酸铵饱和度为85%时比活力最高,达到420.33μmol/min/mg protein,纯化倍数为18.86。根据硫酸铵粗沉淀谷胱甘肽S-转移酶结果,选择硫酸铵浓度为60%～90%沉淀段进行GSH-agarose亲和层析,纯化后比活力最高达到1365.29μmol/min/mg protein,纯化倍数达到61.25。经SDS-PAGE鉴定,得到的GST为1条带,亚基的分子量约为24kDa。     

Partial purification and properties of the Epstein-Barr virus-associated nuclear antigen.

The Epstein-Barr virus (EBV)-associated nuclear antigen (EBNA) was purified 85-fold from a nuclear pellet derived from an EBV-transformed B lyphoblastoid cell line by a five-step procedure consisting of preparation of extract, heating at 80 degrees C in phosphate buffer, ammonium sulfate precipitation, preparative ultracentrifugation, and affinity chromatography on double-stranded DNA-cellulose. The purified complement fixing antigen specifically blocked the anticomplement immunofluorescence assay for EBNA. Several properties indicate a close association of EBNA with chromatin, viz. 1) precipitation of antigenic activity by phosphate buffer and subsequent thermal fractionation; 2) partial sensitivity of antigenic activity to DNase (but not to RNase) and restoration of activity by addition of calf thymus DNA; and 3) specific binding of EBNA to double-stranded DNA-cellulose. Other properties of EBNA, including its unusual heat stability, are described.     

Three phase partitioning as a novel method for purification of ragi (Eleusine coracana) bifunctional amylase/protease inhibitor

The technique of three-phase partitioning (TPP) was used to purify a bifunctional amylase/protease inhibitor from ragi (Eleusine coracana). This process of purification is a potential method used for separation of proteins directly from large volumes of crude suspension. It involves the addition of a salt (ammonium sulphate) to the crude extract followed by the addition of an organic solvent (t-butanol). The addition of t-butanol, in the presence of ammonium sulphate pushes the protein out of the solution to form an interfacial precipitate layer between the lower aqueous and upper organic layers. The process was carried out in two steps. The various conditions required for attaining efficient purification of the protein fractions were optimized. It was seen that 30% ammonium sulphate saturation with 1:1 ratio of crude extract to tert-butanol gave 8.9- and 8.65-fold purification with 83% and 80% yield of amylase inhibitor and trypsin inhibitor, respectively, in step I. In TPP-step II, 60% ammonium sulphate saturation and ratio of aqueous phase to t-butanol of 1:2 gave maximum 20.1- and 16-fold purification with 39.5% and 32% yield of amylase inhibitor and trypsin inhibitor, respectively. The sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the inhibitor protein showed substantial purification and the molecular weight of the protein was found to be 14 kDa.     

Initial characterisation of 4-chlorobenzoate dehalogenase from Pseudomonas sp. CBS3

Extracts of Pseudomonas sp. CBS3 converted 4-chlorobenzoate into 4-hydroxybenzoate. The enzyme responsible for this conversion was enriched by ammonium sulphate fractionation (30–60% saturation, 1.3-fold). The optimum conditions for the reaction were 30–35°C and pH 7–7.5. The enzyme was activated by Mn²⁺ (1 mM final concentration) up to 120-fold, and by Co²⁺ (1 mM final concentration) up to 60-fold. Other divalent ions had no effect. EDTA inhibited the enzyme. 4-Bromobenzoate and 4-iodobenzoate were substrates for the enzyme, but 4-fluorobenzoate was not converted.     

Isolation of antidiabetic components from white-skinned sweet potato (Ipomoea batatas L.)

We have already reported that white-skinned sweet potato (Ipomoea batatas L.) (WSSP) shows antidiabetic activity in streptozotocin (STZ) induced diabetic rats and genetically diabetic models (yellow KK, db/db mice and Zucker fatty rats). In this study, isolation and purification of the antidiabetic component of WSSP were attempted. Almost all antidiabetic activity was found in the cortex of WSSP. The fractionation of the antidiabetic component in the WSSP cortex was done by the following methods: dialysis of the water extract, 85% ethanol precipitation, 15% trichloroacetic acid (TCA) treatment, butyl-, phenyl-hydrophobic column chromatography, and ultrafiltration treatment. The antidiabetic component was not eliminated during dialysis and was soluble in 85% ethanol and 15% TCA, but it passed through a filter that allows the passage of substances of a molescular weight of 30,000. The uniformity of this isolated active component was analyzed using HPLC. A single peak was seen with three different columns (C8 reverse-phase column, anion exchange QA column, and gel filtration column (GFC)), indicating that the component is a uniform substance. The molecular weight of this antidiabetic component was estimated to be 22,000 by GFC analysis. This active component was presumed to be an acidic glycoprotein because it contained protein and sugar and was adsorbed onto the QA column at pH 7.0.     

Three-liquid-phase extraction of diosgenin and steroidal saponins from fermentation of Dioscorea zingibernsis C. H. Wright

Diosgenin is an important starting material in the steroidal hormone industry. The yield of diosgenin obtained from the fermentation of Dioscorea zingibernsis C. H. Wright (DZW) by Trichoderma harzianum is higher than that typically obtained from acid hydrolysis. In this paper, the extraction of steroids in the culture broth was studied. A novel three-liquid-phase system (TLPS) consisted of petroleum ether, ethanol, ammonium sulphate and water was used to separate diosgenin and steroidal saponins in the culture broth. The partition behaviors of various steroidal saponins, diosgenin and glucose were investigated. From this, an optimized TLPS was obtained, which composed of 30% ethanol (w/w), 17% (NH₄)₂SO₄ (w/w) and 40% (w/w) petroleum ether. In the optimized TLPS, almost all of the diosgenin was extracted into the top phase giving a recovery of 97.24%, whereas the steroidal saponins were mainly extracted into the middle phase, with recoveries of zingibernsis newsaponin, deltonin and diosgenin-diglucoside reaching almost 100%. The recoveries of trillin and diosgenin-triglucoside were 96.03% and 98.82%, respectively. Glucose tended to remain in the bottom phase, giving a recovery of 72.01%. The three-liquid-phase extraction (TLPE) successfully resulted in the simultaneous separation of diosgenin, untransformed steroidal saponins and glucose.     

A new biochemical engineering approach to the fractional precipitation of proteins

A biochemical engineering framework for optimizing the design and operation of fractional protein precipitation has been developed. The method utilizes a fractionation diagram to represent the purification of a product protein relative to total contaminating protein. The purification factor for a single or double-cut fractional precipitation is obtained as the gradient of an appropriate operating tie-line. A computer algorithm has been devised to maximize the tie-line gradient for a given yield enabling a plot of optimum purification factor versus yield to be constructed. The recovery of the enzyme alcohol dehydrogenase from clarified bakers homogenate using saturated ammonium sulphate has been examined. Fractionation and purification versus yield diagrams were used to investigate the effects of such process parameters as pH, temperature, and initial total protein concentration on fractionation efficiency. The results are discussed in terms of the underlying solubility and mixing phenomena and the industrial application of fractional precipitation.     

Identification of multiple cell surface antigens associated with the sex pheromone response of Streptococcus faecalis.

High-molecular-weight surface antigens, obtained by ammonium sulfate precipitation of culture supernatants and identified in Western blots of sodium dodecyl sulfate-polyacrylamide gels, have been correlated with the sex pheromone response of Streptococcus faecalis donor cells. Pheromone-induced cells carrying the conjugative plasmid pCF10 produced both an antigenic component (C130) composed of at least four bands in the range of 130 kilodaltons and a 73-kilodalton antigen (SA 73). The concentration of the C130 antigen in culture supernatants increased with time after exposure of donor cells to pheromone preparations. Gel filtration studies indicated that this antigen exists in the native state as a very large complex that is more than 180,000 daltons in size. The C130 antigen was susceptible to digestion by proteinase K and was not reactive with either concanavalin A or wheat germ agglutinin. The antigenicity of C130 was not destroyed by treatment of blots with trypsin, chymotrypsin, or papain before development with antibody, whereas the antigenicity of SA73 was susceptible to these treatments.     

Nitrite oxidase and nitrate reductase in Nitrobacter agilis

Nitrite oxidase and nitrate reductase in Nitrobacter agilis were shown to be separate enzymes. The best separation of the two systems was achieved by ammonium sulphate fractionation. The effects of various compounds, including antimycin A, 2-n-heptyl-4-hydroxyquinoline N-oxide and chlorate, also clearly distinguish between the two enzyme reactions. The relationship between the two opposing reactions in Nitrobacter is discussed.