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.     

Isolation and characterization of a zinc-containing metalloprotease expressed by Vibrio tubiashii

A Vibrio tubiashii hemagglutinin, a protease, was purified by ammonium sulfate precipitation, gel filtration, and hydrophobic interaction chromatography. It agglutinates sheep, chicken, bovine, rabbit, guinea pig, and human erythrocytes. It has a molecular mass of 35 kDa, isoelectric points of 3.5 and 3.7, and is inhibited by ortho-phenanthro line, phosphoramidon, and Zincov. The N-terminal amino acid sequence (Ala-Gln-Ala-Thr-Gly-Thr-Gly- Pro-Gly-Gly-Asn-Gln-Lys-Thr-Gly-Gln- Tyr-Asn-Phe-Gly) has strong homology to other Vibrio proteases.     

Toxoid of Clostridium botulinum type F: purification and immunogenicity studies.

Toxin from Clostridium botulinum type F was recovered from dialysis cultures and partially purifed by: (i) ammonium sulfate and ethanol precipitation; (ii) O-(diethylaminoethyl)-cellulose chromatography; or (iii) diethylaminoethyl-cellulose chromatography followed by O-(carboxymethyl)-cellulose chromatography. Toxin purities as reflected by specific activity were 1.83 X 10(6), 9.8 X 10(6), and 2.0 X 10(7) mouse 50% lethal doses (LD50)/mg of N, respectively, for toxins purified by the three methods. The toxins were converted to toxoids by incubation at 35 C in the presence of 0.3 to 0.45% formalin for 21 to 35 days. Toxoids were immunogenic in guinea pigs, as demonstrated by serum antitoxin response and the immunized animals' resistance to challenge by type F botulinal toxin. The immune response to type F toxoids was lower when toxoids of serotypes A, B, C, D, and E were combined with the type F toxoid than when the type F toxoid only was administered. The toxoid prepared from the most highly purified toxin (method [iii]) conferred the highest immunity in guinea pigs at a given dose level. A relation between serum antitoxin level and resistance to challenge was observed. At least 50% of the groups of guinea pigs with 0.015 antitoxin units or more per ml survived challenge by 10(5) mouse LD50 of type F botulinal toxin. A dose of 3.75 mug of N of the most highly purified type F toxoid in combination with the other five serotypes of botulinal toxoid invoked an immune response in guinea pigs comparable to that considered adequate for the other toxoids.     

Precipitation Reactions in Agar Between Swine Serum and Homologous Pancreas Extracts

Hyperimmune anti-hog cholera and nonimmune swine sera yielded approximately 50% more precipitation reactions in agar-gel diffusion tests with pancreas extracts from SPF noninfected swine than with extracts obtained from swine experimentally infected with virulent hog cholera virus. The pancreas-reacting property of swine serum was determined to be relatively heat stable, withstanding 68 C for 30 minutes. Of various swine serum fractions tested, the only one that reacted with pancreas extracts contained gamma, beta and alpha-globulins. In the absence of alpha-globulin, precipitation reactions were not observed. Sera of newborn SPF piglets, containing 50% alpha-2 globulin, formed more intense precipitation lines with swine pancreas extracts than were formed by the sera of their dams with the same extracts. The pancreas-reacting activity of swine sera was completely removed by absorption with pancreatic tissue. This property was not removed by absorption with guinea pig kidney, or beef, swine or human erythrocytes. Maceration of pancreatic tissue released reactive substances in a polydispersed form. This was demonstrated by the ability of almost all supernates and sediments from differential centrifugation of such preparations to form precipitation lines with swine sera. Reactive substances from swine pancreas were found to be relatively heat labile, being inactivated in one hour at 56C. No evidence was obtained in this study to indicate that the observed precipitation reactions were related to hog cholera virus and its corresponding antibody. The reactions are believed to have resulted from the interaction of protein-related substances present in normal swine pancreas with a relatively heat stable component, possibly alpha-globulin, in swine serum.     

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.     

羊抗人IgG的纯化及其在抗—HCV检测中的应用

单独或联合应用辛酸沉淀、饱和硫酸铵沉淀、阴离子交换等方法对羊抗人IgG进行纯化,对纯化前后抗体的纯度和免疫学活性进行比较,并与辣根过氧化物酶连接,作为二抗用于抗－HCV的ELISA检测。结果表明,不同方法纯化的抗体其纯度和免疫学活性具有一定程度的差别,其中经辛酸＋饱和硫酸铵沉淀纯化的抗体为最佳,凝胶扫描纯度为98．05％,比活性近1800,为纯化前的6．8倍。用痞根过氧化物酶标记后,作为酶标二抗检测HCV阴性和阳性标准血清各40份,阴性符合率为97．5％,阳性符合率为95％,可用于抗－HCV的ELISA检测。     

Two pathways in the biosynthesis of cadystins (gamma EC)nG in the cell-free system of the fission yeast

Small metal-binding peptides, cadystins, with the general structure of (gamma-Glu-Cys)n-Gly ((gamma EC)nG), were synthesized in a cell-free system of fission yeast to examine the in vivo synthetic pathway. The crude enzyme for cadystin synthesis was prepared by ammonium sulfate precipitation (75% saturation) from the 120,000 x g supernatant of the cell extract, and the excess salt in the enzyme fraction was removed by Sephadex gel filtration. Using this crude enzyme fraction, it was shown that there were two pathways for cadystin biosynthesis. One pathway is gamma-Glu-Cys (gamma EC) dipeptidyl transfer from both glutathione (gamma ECG) and cadystins to glutathione and cadystins. The other one is gamma EC polymerization from (gamma EC)n and glutathione to (gamma EC)n + i, followed by glycine addition with glutathione synthetase.     

A serum factor cross-reactive with antibodies to a determinant of rabbit encephalitogenic sequence 65–74 of myelin basic protein

A serum factor. cross-reactive with antibodies to a defined determinant of myelin basic protein (residues 66–71), has been found in the sera of nine mammalian species where it may function as a specific neuroautotolerogen. In equilibrium competitive inhibition radioimmunoassays the factor appears to be completely competitive with synthetic peptide S24 (TTHYGSLPQKG) at high affinity and is therefore termed MBP-SF-24 (myelin basic protein serum factor of the S24 type). The bulk of the activity can be recovered by ammonium sulfate fractionation at 61.1% saturated ammonium sulfate (SAS), pH 7, (fractionE) after removal by precipitation at pH 7 of the 37.5, 42.6, 47.5, and 51.4% SAS fractions (fractionsA-D), including the immunoglobulins, and before removal by precipitation at pH 5 of the albumin fraction (fractionF). The factor, by its retention on XM300 during ultrafiltration of fractionE, can be purified 20-fold from serum proteins without much loss through a combination of SAS fractionation and ultrafiltration. The yield of MBP-SF-S24 in fractionE may range from a low 26 pmol S24 equivalents from 10 ml in sheep serum to a high 1.7 nmoles from 10 ml rat serum. The serum factor is reactive at high affinity with each of two populations of S24-reactive antibodies in one rabbit reagent antiserum and with one of two populations of S24-reactive antibodies in another. It appears to express a determinant involving residues THYGSL (66–71) of myelin basic protein with the same conformation as found in intact S24.This work was supported at Duke University Medical Center by Research Grant NS-10237 from the National Institutes of Health of the U. S. Public Health Service and the Medical Scientist Training Program Grant #5-T32-OMO-7171-08; at St. Luke's Hospital Center by RG-1197-B-6 from the National Multiple Sclerosis Society and by a grant from the M.T. Biddle Foundation; and at Northwestern University by Research Grant NS-06262 from the National Institutes of Health of the U.S. Public Health Service.     

IMMUNOLOGICAL STUDIES ON PEPSIN AND PEPSINOGEN

1. Alkali (pH 7.6)-denatured pepsins from swine, cattle, and guinea pigs precipitate in swine pepsin antiserum. Similarly treated pepsins from the rabbit, chicken, and shark do not. 2. Pepsin antisera react with both pepsin and pepsinogen, but do not react with the serum proteins from the homologous species. 3. Pepsinogen antisera react with pepsinogen, but not with twice crystallized pepsin, nor with the serum proteins from the homologous species. Positive reactions between activated pepsinogen and pepsinogen antiserum have been observed. It was possible to remove the reacting material from either the pepsinogen or the activated pepsinogen mixture. 4. Antisera made with serum proteins do not react with the homologous pepsin or pepsinogen.     

Efficient recovery of recombinant human erythropoietin from milk of transgenic pigs by two-step pretreatment

The selective removal of impurity proteins and colloidal particles from milk prior to chromatographic purification processes presents a crucial issue in the production of therapeutic proteins from transgenic animals with high recovery yield and purity. We have developed an efficient two-step precipitation method for the recovery of the recombinant human erythropoietin (rhEPO) of interest from transgenic sow milk. Here, rhEPO was partially purified from transgenic sow milk via a two-step precipitation method consisting of ammonium sulfate and divalent metal precipitations, with a yield of approximately 82.1% and a purification fold of 10.4 at a copper concentration of 30 mM. Copper proved to be the strongest flocculating agent among the divalent ions tested for the aggregation of milk proteins under 35%, with ammonium sulfate, zinc, nickel, and calcium demonstrating increasing flocculating capability in the given order. Copper and zinc proved to be appropriate divalent metals for the recovery of rhEPO at high yield and purity, and the optimal concentration ranges of copper and zinc were 20~40 and 40~80 mM, respectively.     

Energy enzymes from the flight muscle of the house sparrow, Passer domesticus-II. Physical and chemical properties related to purification of the glycolytic enzymes

The isoelectric points (pI0), molecular weights and ammonium sulfate precipitation ranges for most of the glycolytic enzymes from house sparrow (Passer domesticus) flight muscle were determined. The pI0 for each enzyme is as follows: HK (6.8), PGI (6.7), PFK (5.4), Ald (7.2), TPI (7.5), PGK (7.1), PGM (6.1), Enol (6.2), PyK (6.6), and LDH (8.3). The molecular weight for each enzyme is as follows: PGI (145,000), Ald (160,000), TPI (60,000), PGK (35,000), PGM (60,000), Enol (100,000), PyK (200,000), and LDH (145,000). The ammonium sulfate precipitation range for each enzyme is as follows: PGI (0-80%), PFK (40-50%), Ald (40-65%), TPI (30-90%), PGK (70-90%), PGM (30-80%), Enol (45-80%), PyK (55-85%), and LDH (40-65%).     

Ammonium sulfate precipitation of the glucocorticoid receptor from rat liver

The transformed glucocorticoid receptor (GR) from rat liver precipitated at 30% saturation of ammonium sulfate and sedimented at 4.3 S on glycerol gradient centrifugation, whereas the nontransformed GR precipitated at higher concentrations of ammonium sulfate (40-50% saturation) and sedimented at 8.6 S on a gradient. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that heat shock protein 90 (hsp 90) precipitated at 40-50% saturation of ammonium sulfate. Moreover, hsp 90 and the nontransformed GR were eluted from DEAE high performance ion-exchange chromatography at similar salt concentrations (0.22-0.23 M NaCl), whereas the transformed GR was eluted at 0.1 M NaCl. Therefore, hsp 90 seems to be responsible for the surface charge characteristics of the nontransformed GR.     

The enzymatic conversion of arachidonic acid into 8,11,12-trihydroxyeicosatrienoic acid. Resolution of rat lung enzyme into two active fractions

The conversion of arachidonic acid into 8,11,12-trihydroxyeicosatrienoic acid by rat lung high-speed supernatant has been resolved into two separate stages through ammonium sulfate precipitation. The first stage is catalysed by 0-30% ammonium sulfate fraction and converts arachidonic acid and 12-hydroperoxyeicosatetraenoic acid into an intermediate, X. X is subsequently utilized in the second stage by the fraction sedimented at 30-50% saturation in ammonium sulfate to form two isomeric 8,11,12-trihydroxyeicosatrienoic acids.     

Hemoglobin & serum albumin: salt-mediated hydrophobic chromatography.

The binding of pure human serum albumin and pure human hemoglobin to L-phenylalinine Sepharose and aniline Sepharose columns, two chromatographic columns of differing hydrophobicity, has been investigated for various concentrations of ammonium sulfate salts.The binding of hemoglobin at a lower ammonium sulfate concentration than albumin in both hydrophobic support systems parallels the solubility and precipitation characteristics of these two proteins in solution and mirrors the phenomenon of salting out of proteins in solution. Both hemoglobin and albumin bind at lower concentrations on aniline Sepharose than on L-phenylalanine Sepharose, reflecting the greater efficiency of binding by the more hydrophobic support matrix.     

Protein kinase activities in mammalian blood fluid

Two protein kinase activities, one specific for phosvitin and another specific for histone, were detected in serum and plasma of calf as well as of human blood after precipitation with ammonium sulfate (40%) and chromatography on DEAE-Sephacel. The enzymes were separated by chromatography on phosphocellulose. The histone kinase is not related to the cyclic AMP-dependent protein kinase; it may derive at least partly from damaged cells. The phosvitin kinase activity carries characteristics of the so called casein kinase type II similar to that present at the surface of cells including blood cells.     

Improved method for purification of enterotoxin from Clostridium perfringens type A.

The purification procedure of Clostridium perfringens type A enterotoxin has been improved. The cell sonic extract was precipitated twice with ammonium sulfate, first 40% saturated to concentrate the enterotoxin and then 15% saturated. The two precipitations were followed by gel filtration on Sephadex G-100. The enterotoxin appeared to be homogeneous on 7% polyacrylamide gel electrophoresis after this three-step purification procedure, with a recovery of 56% and a 12.3-fold purification. The solubility properties at different pH values, temperatures, and ammonium sulfate concentrations are also given as basis for the purification procedure.     

Improved method for purification of enterotoxin from Clostridium perfringens type A

The purification procedure of Clostridium perfringens type A enterotoxin has been improved. The cell sonic extract was precipitated twice with ammonium sulfate, first 40% saturated to concentrate the enterotoxin and then 15% saturated. The two precipitations were followed by gel filtration on Sephadex G-100. The enterotoxin appeared to be homogeneous on 7% polyacrylamide gel electrophoresis after this three-step purification procedure, with a recovery of 56% and a 12.3-fold purification. The solubility properties at different pH values, temperatures, and ammonium sulfate concentrations are also given as basis for the purification procedure.     

Studies on Complement-Fixation Reaction in Equine Infectious Anemia

Complement-fixing (CF) and complement fixation-inhibiting (CFI) antibodies were investigated sequentially in three horses infected with equine infectious anemia (EIA) virus. The CF activity was first demonstrated 6 or 8 days after onset of the first pyrexia. The CFI activity developed a short period later, and caused a decrease of apparent CF titer of the whole serum. However, both antibodies tended to increase with advance of the disease course in two persistently infected horses, whereas they became completely undetectable during the late-stage in the remaining horse which showed no evidence for recurrence of pyrexia or persistence of viremia. The CF activities determined with varying dilutions of serum were distinctly different in pattern between the early-stage serum having the CF activity alone and the late-stage serum having both the CF and the CFI activities. The CF antibody was precipitated by 27–30% saturation with ammonium sulfate (SAS) while the CFI activity distributed in a wider range of precipitates formed by 26–32% SAS. The CFI activity was demonstrated to a higher titer when a relatively small amount of antigen was sensitized with CFI antibody prior to addition of reference CF antibody than when the three reagents were mixed at one time. The late-stage serum with a strong CFI activity against EIA antigen had no effect on the CF reaction of other viral antigen–antibody systems such as equine influenza and equine rhinopneumonitis.     

Bactericidal activity of human, swine and cattle serum against pseudomonas aeruginosa strains

The aim of this study was to assess of bactericidal activity of human, swine and cattle serum against 136 of Pseudomonas aeruginosa strains isolated from people, fishes, domestic and fur animals. The mechanism of the bactericidal activity of serum against gram-negative bacteria is complex and involves the participation of complement, antibodies and lysozyme (1, 5, 7, 8, 10, 11, 13, 14, 24, 25, 27, 30). The susceptibility of gram-negative rods to serum is differentiated. Pseudomonas aeruginosa strains are the most resistant (17, 25, 30). This opportunistic pathogen produce proteases that destroy complement components and immunoglobulins (3, 18, 19). The bactericidal activity of serum was determined after 3 hours incubation of bacteria in 50% serum by the method of Jankowski (1981) (5). The results of this study indicate that 71% of this strains were resistant to swine serum action, 68% of this strains were resistant to bovine serum and 57% of the Pseudomonas aeruginosa strains were sensitive to human serum. The P. aeruginosa strains isolated from fishes were the most sensitive to serum action and the strains isolated from people and cattle were most resistant to the bactericidal activity of serum.     

Identification of ovotransferrin as a heme-, colony- and burst-stimulating factor in chick erythroid cell cultures

Clonal growth of erythroid cells from bone marrow of 2-day-old chicks in fibrin clots under different culture conditions has been used as independent assays for heme-, colony- and burst-stimulating activities found in anemic chicken plasma. The properties of the heme-stimulating activity analyzed by gel filtration, isoelectrofocusing, resistance to heat denaturation, ammonium sulfate precipitation, DEAE-chromatography or HAP-chromatography, suggested serum transferrin as the heme-stimulating factor(s). Heme-, colony- and burst-stimulating factor(s) from anemic chicken plasma did not separate from each other by gel filtration or isoelectrofocusing. Ovotransferrin from egg white also showed heme-, colony- and burst-stimulating activities by the assay employed even after further purification by limited trypsin digestion, electrophoresis, hydroxylapatite chromatography or fractionation by ConA-Sepharose chromatography.