PURIFICATION AND CRYSTALLIZATION OF DIPHTHERIA ANTITOXIN

Purified preparations of diphtheria antitoxin have been obtained by digestion of the toxin-antitoxin complex with trypsin, followed by fractional precipitation with ammonium sulfate. The various fractions obtained in this way are all 90 per cent or more precipitated by diphtheria toxin but combine with different quantities of the toxin. The fraction precipitated between 0.33 and 0.5 saturated ammonium sulfate is homogeneous by electrophoresis and ultracentrifuge but does not have constant solubility. A small amount of a more soluble fraction has been obtained which does have constant solubility and satisfies the criteria of a pure protein. This protein crystallizes readily in poorly formed thin plates. It is very unstable and reverts to a less soluble non-crystallizable form. It has a sedimentation constant of 5.7 x 10^–13 and a molecular weight of 90,500. It has an antitoxic value of 700–900 flocculation units per mg. protein nitrogen and has an antitoxic value by the protection test of about 700 units per mg. protein nitrogen. The precipitation range of the purified antitoxin with purified toxin is much wider than that with crude preparations.     

Quantitative comparison on the refinement of horse antivenom by salt fractionation and ion-exchange chromatography

A quantitative comparison was made on the fractionation of pepsin-digested horse antivenoms by ammonium sulfate (AS) fractional precipitation and ion-exchange chromatography on Q-Sepharose. In the precipitation process, pepsin digested horse anti-Naja kaouthia serum was precipitated by 30% saturated AS followed by 50% saturated AS. The recovery of antibody activity [as measured by an enzyme-linked immunosorbent assay (ELISA) against the cobra postsynaptic neurotoxin 3] from the 30–50% saturated AS precipitate was 53% with a 1.93-fold purification. For the chromatographic process, the behavior of the horse antitoxin antibody and its F(ab′)₂ fragments was first studied. The pepsin digested horse serum was then desalted on a Bio-gel P-2 column followed by chromatography on Q-Sepharose using a linear gradient (20 mM Tris-HCl, pH 8.0 containing 0.0 to 0.5 M NaCl). A peak containing primarily the F(ab′)₂ antibody could be obtained. This peak constituted 73% of the total antivenom activity with 2.08-fold purification. The total recovery of antibody activity by the chromatographic process was 90%. The yield of antibody activity was about 2-fold higher than that reported previously with other fractionation procedures. The implications of these results for the refining of horse therapeutic antivenoms are discussed.     

Determination of the Optimal Ammonium Sulfate Concentration for the Fractionation of Rabbit, Sheep, Horse, and Goat Antisera

Various ammonium sulfate concentrations and reaction conditions were employed in the fractionation of sera from rabbits, sheep, horses, and goats. Precipitates and supernatant fluids were analyzed by electrophoresis to study the effects of the controlled variables. At room temperature, the third precipitate in 35% saturated (NH(4))(2)SO(4) was the best fraction from both rabbit and sheep sera; 80 to 90% of the gamma globulins were recovered. The second and third precipitates of horse sera proteins in 30% saturated (NH(4))(2)SO(4) were both satisfactory, but only 44% of the gamma globulin was recovered after three precipitations. Goat sera yielded a very satisfactory fraction; 80% of the gamma globulin was recovered after two precipitations-the first in 30% and the second in 45% saturated (NH(4))(2)SO(4). The composition of these fractions was not influenced by the pH of the sulfate solutions (pH 5.8 and 7.2), by a range of normal room temperatures (20 to 30 C), or by diluting the sera before fractionation. Crude globulins and fluorescein isothiocyanate-labeled globulins were successfully refractionated by one precipitation in the optimal sulfate concentration for the appropriate animal species. The refractionated products contained considerably less beta and alpha globulins than did the original crude fractions and little or no albumin.     

Alzheimer disease paired helical filament core structures contain glycolipid.

The core structures of sodium dodecyl sulfate extracted, pronase digested paired helical filaments of Alzheimer disease were solubilized by heating in dimethyl sulfoxide. Electron microscopy revealed that after heating in dimethyl sulfoxide, intact paired helical filaments were no longer present in the dimethyl sulfoxide soluble fractions or in the insoluble lipofuscin-containing fractions. Enzyme-linked immunosorbent assays of the various fractions with the monospecific antibody A128 to paired helical filaments demonstrated 96% of the immunoreactivity to be in the dimethyl sulfoxide soluble fraction, and only 4% in the dimethyl sulfoxide insoluble fractions. Lyophilization of the dimethyl sulfoxide soluble supernatant and resuspension in water failed to reassociate the paired helical filaments, but did result in an insoluble precipitate. Analysis of the dimethyl sulfoxide solubilized paired helical filament fraction by nuclear magnetic resonance revealed it to be composed of glycolipid in a form that was distinct from similar fractions isolated from normal aged control brains. The aggregation of an altered glycolipid to form paired helical filaments in Alzheimer disease could explain their insolubility.     

Further studies on cold insoluble circulating immune complexes in rabbits immunized with bovine albumin.

Cold insoluble circulating immune complexes of BSA and anti-BSA antibody are formed in vivo while immune catabolism of antigen is occurring. The effects of temperature, rate of precipitation and redissolvability of the cold insoluble antigen were studied in this model. Circulating BSA is soluble at 37 degrees, but may precipitate when the temperature is reduced. The solubility of antigen decreases at 24 degrees and below. Complete precipitation occurs in 5-7 days. The antigen in the cryoprecipitate is difficult to redissolve unless low pH citrate or glycine buffers are used.     

In vitro recognition of alloantigens: nature of responding and stimulating cells.

Immunosuppressive and immunostimulatory activity of human cancer ascitic fluids has been examined using the in vitro primary plaque-forming cell response (PFR) to sheep erythrocytes (Mishell-Dutton assay). We have prepared three fractions of ascitic fluid by precipitation with ammonium sulfate. Suppressive activity occurred in the fraction which was insoluble at 50% of saturation but not those fractions which precipitated at 30 or 80% of saturation. The fractions which were precipitated at 30 and 80% were stimulatory in the assay system. Normal human serum also had suppressive activity in the fraction precipitated at 50% of saturation but not as much as was found in ascitic fluid. Serum did not yield any fractions with stimulatory activity.     

Antibody binding capacity of different peptide chains isolated from digested and purified horse diphtheria antitoxin

In commercial digested and purified horse diphtheria antitoxin, which is formed largely of the gamma globulin fragment with the sedimentation coefficient 5.3 S, the reactive disulphide bonds were destroyed by S-sulphonation. Gel filtration on Sephadex G-100 in 0.05 M formic acid with 6 M urea showed that the molecule of the S-sulphonated preparation dissociated into chains similar in character to the peptide chains of native horse antitoxins. Antibody activity was still partly maintained even after treatment with 6 M urea. On mixing the two types of chains isolated by gel filtration, antibody activity was recovered, the amount of antibody protein determined in the mixed fractions being greater than the sum of the amounts in the separate fractions. The neutralizing activity of the mixed fractions tested against toxinin vivo was also greater than the sum of the activity of the separate fractions.     

Lymphocyte cytotoxicity in x-irradiation-induced rat small bowel adenocarcinoma. III. Blocking by 3 M KCL extract

Hypertonic salt extracts (3 M KCl) of x-irradiation-induced Holtzman rat small bowel adenocarcinomas blocked the in vitro destruction of allogeneic cultured cells of this malignancy by sensitized lymphoid cells obtained from tumor-bearing animals. The protective effect were mediated by a blocking action at both the effector and the target cell level. The extracts were separated into 50% ammonium sulfate soluble and insoluble fractions with the soluble fraction being more effective in blocking the cytotoxic responses through interaction with the lymphoid cells whereas the insoluble one had a greater effect upon tumor target cells. Associated with both fractions was the oncofetal glycoprotein previously identified with the cellular membrane of this x-ray-induced malignancy. Immunoglobulins were identified with insoluble fraction; some were able to bind the oncofetal protein, thus clasifying it as a fetal antigen. The protective effects of the soluble fraction and this neoantigen were found to be citric acid labile, whereas the effects due to the insoluble fraction were unchanged.     

Separation and characterization of the soluble and insoluble components of insoluble laminaran

Insoluble laminaran, a (1→3)-β-D-glucan from Laminaria hyperborea (L. cloustoni), has been fractionated by differential solubility into soluble and insoluble fractions. These fractions were degraded with a purified exo-(1→3)-β-D-glucanase from Basidiomycete sp. QM806 giving, as primary hydrolysis products, D-glucose, gentiobiose, laminarabiose, and 1-O-β-laminarabiosylmannitol. Gentiobiose was obtained in only trace amounts from the insoluble fraction of laminaran, suggesting the absence of branching. Successive application of periodate oxidation, reduction, mild acid hydrolysis, and enzymic degradation indicated that the branch in the soluble fraction consists of a single β-(1→6)-linked D-glucosyl residue. The results indicate that “insoluble” laminaran is apparently an aggregate of three closely related polysaccharide species: a soluble, branched, reducing component (soluble laminarose); an insoluble, unbranched, reducing component (insoluble laminarose); and an unbranched, nonreducing component (laminaritol) that has a monosubstituted mannitol residue at the reducing terminal. Laminaritol was found to be about equally distributed between the soluble and insoluble fractions. The average d.p. of the laminaran components is 20–25 residues, as determined from the relative amounts of enzymic hydrolysis products and from periodate-oxidation data.     

Straightforward isolation of phosphatidyl-ethanolamine-binding protein-1 (PEBP-1) and ubiquitin from bovine testis by hydrophobic-interaction chromatography (HIC)

Isolation of phosphatidyl-ethanolamine-binding protein-1 (PEBP-1) from bovine brain was described almost three decades ago but it required a large number of steps to reach high purity. After the fractionation of bovine testis proteins by ammonium sulfate precipitation we found that PEBP-1, detected by Western blotting, was among the very few proteins still soluble at 80% ammonium sulfate saturation (3.2M). This soluble fraction (S80) was directly loaded onto a phenyl sepharose column equilibrated at the same ammonium sulfate concentration (3.2M). A stepwise elution of the retained material at 1.0, 0.5, 0.2, 0.1M ammonium sulfate in ammonium hydrogen carbonate was performed and then with ammonium hydrogen carbonate alone and finally with 50% ethylene glycol. All fractions were analyzed by SDS-PAGE and Western blotting and the fractions containing PEBP-1 was further fractionated by size exclusion chromatography on a HR75 Superdex column permitting the isolation of ubiquitin in addition to PEBP-1 as demonstrated by Western blotting and mass spectrometry. This study shows the feasibility of hydrophobic interaction chromatography (HIC) on phenyl sepharose at a very high ammonium sulfate concentration (3.2M; 80% saturation) to efficiently purify the proteins that are still soluble in these extreme conditions.     

A simple one-column procedure for the separation of swine and human serum transferrins

A rapid method for the separation of transferrin from swine or human serum is described. Serum (human or swine) is brought to 50% of saturation with ammonium sulfate for removal of immunoglobulins, the resulting precipitate discarded and the supernatant brought to 70% of saturation. The resulting precipitate was dissolved in and dialyzed against 1.54 mM sodium azide (I = 0.00154). Chromatography of the low ionic strength ammonium sulfate fractions (= 20 ml of swine or human serum, 70% of saturation) on columns of Bio-Gel A-1.5 m-Reactive Blue 2, equilibrated with 1.54 mM sodium azide, resulted in two peaks, a breakthrough peak and pure transferrin which was eluted with a linear gradient with 0.5 M potassium phosphate buffer, pH 7.1, as limit buffer. Yields varied between 53 and 55% from whole serum and 70-76% from the ammonium sulfate fractions. Transferrins from both species were found to be homogeneous when subjected to immunoelectrophoresis (anti whole serum antibody) and anionic and sodium dodecyl sulfate polyacrylamide disc gel electrophoresis. Hemopexin, a frequently found contaminant in transferrin preparations, is tightly bound by the gel-dye complex under the experimental conditions. Swine serum transferrin possesses many physicochemical properties practically identical to the human protein. Although small differences in physicochemical properties were apparent the extinction coefficients, molecular weights, electrophoretic mobilities, absorbance maxima of the diferric proteins (470 nm), isoelectric points and the absorbance ratios (465 nm/410 nm) of the diferric proteins were practically identical. Both swine and human transferrin produced a reaction of identity (complete coalescence) when reacted with antibody to either transferrin.     

Isolation and partial characterization of apiogalacturonans from the cell wall of Lemna minor

1. A mild, reproducible extraction procedure, using 0.5% ammonium oxalate, was developed for the isolation of polysaccharides containing d-apiose from the cell wall of Lemna minor. On a dry-weight basis the polysaccharide fractions extracted with ammonium oxalate made up 14% of the material designated cell walls and contained 20% of the d-apiose originally present in the cell walls. The cell walls, as isolated, contained 83% of the d-apiose present in L. minor. 2. After extraction with ammonium oxalate, purified polysaccharides were obtained by DEAE-Sephadex column chromatography and by fractional precipitation with sodium chloride. With these procedures the material extracted at 22 degrees C could be separated into at least five polysaccharides. On a dry-weight basis two of these polysaccharides made up more than 50% of the material extracted at 22 degrees C. There was a direct relationship between the d-apiose content of the polysaccharides and their solubility in sodium chloride solutions; those of highest d-apiose content were most soluble. 3. All the polysaccharides isolated appeared to be of one general type, namely galacturonans to which were attached side chains containing d-apiose. The d-apiose content of the apiogalacturonans varied from 7.9 to 38.1%. The content of esterified d-galacturonic acid residues in all apiogalacturonans was low, being in the range 1.0-3.5%. Hydrolysis of a representative apiogalacturonan with dilute acid resulted in the complete removal of the d-apiose with little or no degradation of the galacturonan portion. 4. Treatment of polysaccharide fractions with pectinase established that those of high d-apiose content and soluble in m-sodium chloride were not degraded, whereas those of low d-apiose content and insoluble in m-sodium chloride were extensively degraded. When the d-apiose was removed from a typical pectinase-resistant polysaccharide, the remainder of the polysaccharide was readily degraded by this enzyme. 5. Periodate oxidation of representative polysaccharide fractions and apiogalacturonans and determination of the formaldehyde released showed that about 50% of the d-apiose molecules were substituted at either the 3- or the 3'-position.     

鲫鱼血清和皮肤粘液IgM的分离纯化及部分性质的鉴定

采用盐析法结合葡聚糖凝胶柱 ,分离纯化鲫鱼血清IgM ;然后制备兔抗鲫鱼血清IgM多克隆抗体 ,将其偶联到Sepharose 4B上制成亲和柱 ,用于分离纯化皮肤粘液IgM。结果表明 :33%～ 4 5 %硫酸铵溶液沉淀处理可以去除鲫鱼血清中除IgM外的很多杂蛋白 ,再经葡聚糖凝胶柱纯化 ,IgM纯度可达 80 %以上 ,其重链和轻链的分子量分别为 79和 2 5kDa ;以兔抗鲫鱼血清IgM多克隆抗体亲和柱分离皮肤粘液IgM ,分离效果良好 ,IgM重链的分子量为 88kDa ;Westernblot显示兔抗鲫鱼血清IgM多克隆抗体识别的是血清和皮肤粘液IgM的重链部分。用ELISA测定鲫鱼血清中IgM含量在一年中的变化 ,结果表明IgM在春夏季的含量高于秋冬季     

IMMUNOCHEMICAL PROPERTIES OF NATIVE AND DENATURED HORSE SERUM GLOBULINS

1. The influence of guanidine hydrochloride on the denaturation and regeneration of Type I antipneumococcal horse serum globulin was determined by measurements of viscosity, diffusion, and sedimentation in the ultracentrifuge. In addition, the effect of NaCNS on the antibody globulin was studied. 2. Both the irreversibly denatured and the regenerated fractions were found to be precipitable by SI. The observed changes in combining ratio have been tentatively explained in terms of (a) changes in the mean molecular weight, or alternatively (b) an increase in the number of serologically active groups upon denaturation, followed by masking of the latter upon regeneration. Discounting a specific effect of NaCNS on either fraction, the extent of specific precipitation is of the same order of magnitude for native and irreversibly denatured antibody. 3. Quantitative precipitin titrations have been performed on rabbit antisera to native and irreversibly denatured horse antibody, and normal globulin GI, respectively. No significant differences in the antigenic activity of these proteins were found. Measurements of their cross-reactivity led to the conclusion that the native and irreversibly denatured fractions of antibody globulin are antigenically more closely related to each other than to the corresponding fractions of normal globulin, and vice versa.     

Failure of the usual anti-Ig antibody method for quantitative measurement of low affinity antibodies

The usual anti-Ig antibody method, consisting of the precipitation of soluble antigen-antibody complexes by heterologous anti-Ig antibody, was applied for quantitative estimation of guinea pig IgG2 anti-ovalbumin and anti-2,4-dinitrophenyl (DNP) antibodies by measuring the maximum amounts of antibody-bound antigens. However, the amounts of antibodies estimated were less than those obtained by other methods: the precipitin reaction, the precipitation of antigen-antibody complexes with 50% saturated ammonium sulfate, and equilibrium dialysis. In particular, the anti-Ig antibody method greatly underestimated the amount of anti-DNP antibody with low affinity for epsilon-DNP-L-lysine. Thus, it was concluded that partial dissociation of the antigen-antibody complexes occurring upon precipitation with anti-Ig antibody made the anti-Ig antibody method unsuitable for quantitative determination of antibodies.     

Carrageenans from chilean samples of Stenogramme interrupta (Phyllophoraceae): structural analysis and biological activity

Carrageenans extracted from cystocarpic and tetrasporic Stenogramme interrupta were analysed by chemical and spectroscopic methods. The carrageenan from cystocarpic plants is composed predominantly of 0.5 M KCl-insoluble and 1 M KCl-soluble fractions. The insoluble fraction contained iota-carrageenan as the major component with alpha-carrageenan and pyruvated carrageenan as minor components. The soluble fraction is highly heterogeneous and did not contain the precursors mu- and nu-carrageenans. The polysaccharide from tetrasporic plants is composed of zeta- and lambda-carrageenans, and low sulfated galactans. It is soluble in KCl and partly cyclized by alkaline treatment. The antiviral and anticoagulant properties of the insoluble polysaccharide fraction from cystocarpic S. interrupta and the polysaccharide from tetrasporic S. interrupta are reported the results of which suggest promising antiherpetic activity.     

The kinetics of protein precipitation by different reagents

The kinetics of precipitation of soya protein has been examined in a tubular flow reactor with the precipitants, ammonium sulfate, ethanol, divalent calcium, and sulfuric acid. The precipitate growth profiles obtained in all cases showed the rapid formation of a precipitate phase and the attainment of a final size within 16 s. The final mean particle diameter d(m) varied with precipitating agent used in the order: sulfuric acid (12.5 mum) > ethanol (7.5 mum) approximately calcium ion (7.2 mum) > ammonium sulfate (3.1 mum). In the case of ethanol precipitation, changes in the design of the contacting section of the reactor led to differences in the precipitate growth curve. Protein solubility curves are also presented, and with the reactor data, they provide a convenient method for assessing the effect of precipitating agent on the design of protein precipitation reactors.     

SOLUBILITY STUDIES ON PURIFIED TOBACCO MOSAIC VIRUS

Different samples of purified tobacco mosaic virus show a relatively wide variation in solubility in ammonium sulfate solution. This variation and the type of solubility curve obtained in the presence of varying amounts of solid phase show that the purified virus whether isolated by mild treatment with ammonium sulfate or by ultracentrifugation is not a homogeneous chemical substance but contains more soluble and less soluble virus fractions of comparable specific activities. Long contact with strong ammonium sulfate solutions or 0.1 M phosphate buffer results in a decrease in solubility. The variation in the solubility of samples isolated from different plants by the same method seems to depend in part on the length of time the plants are inoculated before they are cut, and probably also on the conditions under which they are grown. Virus preparations isolated from plants of different genera grown under the same conditions and inoculated at the same time, however, behaved like identical substances in solubility experiments.     

A (1-->3)-beta-d-Glucan Isolated From Zea Shoot Cell Wall Preparations

A small quantity of (1→3)-β-d-glucan was extracted with a (1→3),(1→4)-β-d-glucan by hot water after treatment of the insoluble fraction of a buffer homogenate of Zea shoots with 3 molar LiCl. An ammonium sulfate precipitation procedure effected a separation of the (1→3)-β-d-glucan from the more prevalent (1→3),(1→4)-β-d-glucan. The minor component polysaccharide precipitated at a concentration of 20% ammonium sulfate (w/v) and was, as a consequence of precipitation, rendered insoluble in water. The insoluble products were dissolved in 1 normal NaOH followed by neutralization with CH₃COOH. The purified polysaccharide accounted for approximately 0.3% of total hot water extract. It consisted mostly of glucose and its average mol wt was estimated to be about 7.0 × 10⁴, based on elution from a calibrated Sepharose CL-4B column. Methylation analysis and enzymic hydrolysis or partial acid-hydrolysis of the polysaccharide followed by analysis of the hydrolysate showed that the polysaccharide consisted of (1→3)-β-linked glucose residues.     

Selective precipitation and purification of monovalent proteins using oligovalent ligands and ammonium sulfate

This paper describes a method for the selective precipitation and purification of a monovalent protein (carbonic anhydrase is used as a demonstration) from cellular lysate using ammonium sulfate and oligovalent ligands. The oligovalent ligands induce the formation of protein-ligand aggregates, and at an appropriate concentration of dissolved ammonium sulfate, these complexes precipitate. The purification involves three steps: (i) the removal of high-molecular-weight impurities through the addition of ammonium sulfate to the crude cell lysate; (ii) the introduction of an oligovalent ligand and the selective precipitation of the target protein-ligand aggregates from solution; and (iii) the removal of the oligovalent ligand from the precipitate by dialysis to release the target protein. The increase of mass and volume of the proteins upon aggregate formation reduces their solubility, and results in the selective precipitation of these aggregates. We recovered human carbonic anhydrase, from crude cellular lysate, in 82% yield and 95% purity with a trivalent benzene sulfonamide ligand. This method provides a chromatography-free strategy of purifying monovalent proteins--for which appropriate oligovalent ligands can be synthesized--and combines the selectivity of affinity-based purification with the convenience of salt-induced precipitation.