scholarly journals PURIFIED DIPHTHERIA ANTITOXIN IN THE ULTRACENTRIFUGE AND IN THE ELECTROPHORESIS APPARATUS

1942 ◽  
Vol 25 (3) ◽  
pp. 487-496 ◽  
Author(s):  
Alexandre Rothen
Keyword(s):  
Molecular Weight ◽  
High Activity ◽  
Isoelectric Point ◽  
Diffusion Constant ◽  
Enzymatic Treatment ◽  
Trypsin Digestion ◽  
Globulin Fraction ◽  
Sedimentation Constant ◽  
Diphtheria Antitoxin ◽  
Horse Plasma

Ultracentrifugation studies of diphtheria antitoxin showed that: 1. Purified antitoxin of high activity obtained from horse plasma without enzymatic treatment has exactly the same sedimentation constant as the globulin fraction obtained in a similar way from normal horse plasma s20water = 6.9 x 10–13. 2. Purified antitoxin obtained with trypsin digestion of the toxin-antitoxin complex has a sedimentation constant of s20water = 5.5 ± 0.1 x 10–13, a diffusion constant of D20water = 5.76 x 10–7, and a molecular weight of about 90,000. Electrophoresis experiments demonstrated that: 1. The trypsin-purified antitoxin has an isoelectric point not far from pH 7.0. 2. The reversible spreading noticed at about pH 7.3 cannot be attributed to heterogeneous preparation. 3. The large increase in the γ-globulin fraction occurring during immunization consists either of antitoxin of various degrees of activity or of some inert protein in addition to the antitoxin.

scholarly journals THE LS-ANTIGEN OF VACCINIA

1943 ◽  
Vol 77 (2) ◽  
pp. 155-164 ◽  
Author(s):  
Theodore Shedlovsky ◽  
Alexandre Rothen ◽  
Joseph E. Smadel
Keyword(s):  
Molecular Weight ◽  
Isoelectric Point ◽  
Specific Volume ◽  
Degradation Products ◽  
Diffusion Constant ◽  
Partial Specific Volume ◽  
Analytical Ultracentrifuge ◽  
Axis Ratio ◽  
Ellipsoidal Shape ◽  
Sedimentation Constant

Studies on LS-protein, the soluble double antigen of vaccinia, and on the degradation products L'S and L''S' have been made with electrophoresis and in the analytical ultracentrifuge. LS, which is homogeneous electrically and in the ultracentrifuge, has an isoelectric point at pH 4.8. At 4°C. its partial specific volume is 0.72 cc./gm., and its diffusion constant is 1.50 x 10–7 cm.2/sec. The sedimentation constant is 6.35 at 20°C., the molecular weight is 214,000, and the molecule appears to have an elongated ellipsoidal shape with an axis ratio of 1:20. L'S and L''S' are homogeneous electrically but not in the ultracentrifuge, L''S' being extremely polydisperse.

scholarly journals PURIFICATION AND CRYSTALLIZATION OF DIPHTHERIA ANTITOXIN

1942 ◽  
Vol 25 (3) ◽  
pp. 465-485 ◽  
Author(s):  
John H. Northrop
Keyword(s):  
Molecular Weight ◽  
Ammonium Sulfate ◽  
Diphtheria Toxin ◽  
Soluble Fraction ◽  
Thin Plates ◽  
Fractional Precipitation ◽  
Protein Nitrogen ◽  
Sedimentation Constant ◽  
Diphtheria Antitoxin ◽  
Pure Protein

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.

scholarly journals THE MOLECULAR WEIGHT AND ISOELECTRIC POINT OF THYROGLOBULIN

1935 ◽  
Vol 19 (1) ◽  
pp. 95-108 ◽  
Author(s):  
Michael Heidelberger ◽  
Kai O. Pedersen
Keyword(s):  
Molecular Weight ◽  
Isoelectric Point ◽  
Specific Volume ◽  
Sedimentation Equilibrium ◽  
Sedimentation Constant ◽  
Diffusion Constants ◽  
Equilibrium Data ◽  
Round Numbers ◽  
And Diffusion

1. The sedimentation constant of hog thyroglobulin is 19.2ċ10–13. That of human thyroglobulin is essentially the same. 2. The specific volume of hog thyroglobulin is 0.72. 3. The isoelectric point of native hog thyroglobulin is at pH 4.58, that of denatured thyroglobulin at pH 5.0. 4. The molecular weight of hog thyroglobulin is, in round numbers, 700,000, as calculated from the sedimentation and diffusion constants, or 650,000, as calculated from the sedimentation equilibrium data. 5. The thyroglobulin molecule deviates markedly from the spherical.

NOUVELLES DONNÉES SUR LE POIDS MOLÉCULAIRE DE LA GONADOTROPHINE DU SÉRUM DE JUMENT GRAVIDE

1960 ◽  
Vol XXXV (II) ◽  
pp. 221-224
Author(s):  
Roland Bourrillon ◽  
René Got
Keyword(s):  
Molecular Weight ◽  
Phosphate Buffer ◽  
Diffusion Constant ◽  
Sedimentation Constant

ABSTRACT Sedimentation constant of pregnant mares' serum gonadotrophin remains equal to 3.7 in water, phosphate buffer pH 7.3 and ClNa 0.1 m. But, diffusion constant, equal to 10.2 in water, becomes 4.2 in ClNa 0.1 m. Then, the molecular weight gets 68 500 instead of 28 000 in water.

Cross-Linked αsγt-Chain Hybrids in Plasma Clots Studied by 1D- and 2D Electrophoresis and Western Blotting

1993 ◽  
Vol 70 (03) ◽  
pp. 438-442 ◽  
Author(s):  
B Grøn ◽  
C Filion-Myklebust ◽  
S Bjørnsen ◽  
P Haidaris ◽  
F Brosstad
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibodies ◽  
Human Plasma ◽  
Western Blotting ◽  
Isoelectric Point ◽  
Factor Xiii ◽  
Cross Linking ◽  
2D Electrophoresis ◽  
Complex Phenomenon ◽  
Fibrinopeptide A

SummaryFibrinogen and fibrin related chains in reduced human plasma as well as the bonds interlinking partially cross-linked fibrin from plasma clots have been studied by means of 1D- and 2D electrophoresis and Western blotting. Immunovisualization of reduced plasma or partially cross-linked fibrin with monoclonal antibodies specific for the α-chains or the γ-chains have shown that several bands represent material belonging to both chains. In order to decide whether these bands constitute αγ-chain hybrids or superimposed α- and γ-chain dimers, the cross-linked material was separated according to both isoelectric point (pI) and molecular weight (MW) using Pharmacia’s Multiphor II system. Western blotting of the second dimension gels revealed that partially cross-linked fibrin contains αsγt-chain hybrids and γ- polymers, in addition to the well-known γ-dimers and α-polymers. The main αsγt-chain hybrid has a pI between that of the α- and the γ-chains, a MW of about 200 kDa and contains Aα-chains with intact fibrinopeptide A (FPA). It was also observed that soluble fibrinogen/fibrin complexes as well as partially cross-linked fibrin contain degraded α-dimers with MWs close to the γ-dimers. These findings demonstrate that factor XIII-catalyzed cross-linking of fibrin is a more complex phenomenon than earlier recognized.

scholarly journals CONCENTRATION AND PURIFICATION OF BACTERIOPHAGE

1938 ◽  
Vol 21 (3) ◽  
pp. 335-366 ◽  
Author(s):  
John H. Northrop
Keyword(s):  
Molecular Weight ◽  
Small Molecules ◽  
Specific Activity ◽  
Active Agent ◽  
Pure Substance ◽  
Solubility Curve ◽  
Denatured Protein ◽  
Sedimentation Constant ◽  
Rate Of Sedimentation ◽  
Living Organisms

1. A method for isolating a nucleoprotein from lysed staphylococci culture is described. 2. It is homogeneous in the ultracentrifuge and has a sedimentation constant of 650 x 10–13 cm. dyne–1 sec.–1, corresponding to a molecular weight of about 300,000,000. 3. The diffusion coefficient varies from about 0.001 cm.2/day in solutions containing more than 0.1 mg. protein/ml. to 0.02 in solutions containing less than 0.001 mg. protein/ml. The rate of sedimentation also decreases as the concentration decreases. It is suggested, therefore, that this protein exists in various sized molecules of from 500,000–300,000,000 molecular weight, the proportion of small molecules increasing as the concentration decreases. 4. This protein is very unstable and is denatured by acidity greater than pH 5.0, by temperature over 50°C. for 5 minutes. It is digested by chymo-trypsin but not by trypsin. 5. The loss in activity by heat, acid, and chymo-trypsin digestion is roughly proportional to the amount of denatured protein formed under these conditions. 6. The rate of diffusion of the protein is the same as that of the active agent. 7. The rate of sedimentation of the protein is the same as that of the active agent. 8. The loss in activity when susceptible living or dead bacteria are added to a solution of the protein is proportional to the loss in protein from the solution. Non-susceptible bacteria remove neither protein nor activity. 9. The relative ultraviolet light absorption, as determined directly, agrees with that calculated from Gates' inactivation experiments in the range of 2500–3000 Å. u. but is somewhat greater in the range of 2000–2500 Å. u. 10. Solubility determinations showed that most of the preparations contained at least two proteins, one being probably the denatured form of the other. Two preparations were obtained, however, which had about twice the specific activity of the earlier ones and which gave a solubility curve approximating that of a pure substance. 11. It is suggested that the formation of phage may be more simply explained by analogy with the autocatalytic formation of pepsin and trypsin than by analogy with the far more complicated system of living organisms.

scholarly journals MOLECULAR WEIGHT AND ELECTROPHORESIS OF CRYSTALLINE RIBONUCLEASE

1940 ◽  
Vol 24 (2) ◽  
pp. 203-211 ◽  
Author(s):  
Alexandre Rothen
Keyword(s):  
Molecular Weight ◽  
Isoelectric Point ◽  
Unit Weight ◽  
Diffusion Measurement ◽  
Average Value ◽  
Rate Of Sedimentation ◽  
And Diffusion ◽  
Good Agreement

Electrophoretic studies on purified crystalline ribonuclease showed the absence of any impurities differing in mobility from the bulk of material. The isoelectric point of ribonuclease was found by electrophoresis to be at about pH 7.8. Ultracentrifuge studies indicated fair homogeneity of ribonuclease in solution. Only one moving component has been observed. The molecular weight of ribonuclease was found to be 12,700 from rate of sedimentation (S25 = 1.85 x 10–13 in 0.5 M (NH4)2SO4) and diffusion measurement (D = 1.36 x 10–6 in 0.5 M (NH4)2SO4), in good agreement with the average value of 13,000 found from equilibrium measurements. This low value for the molecular weight of a protein would seem to discredit the value 17,600 as representing a universal unit weight for proteins in general.

scholarly journals ISOLATION, CRYSTALLIZATION, AND PROPERTIES OF PEPSIN INHIBITOR

1941 ◽  
Vol 24 (3) ◽  
pp. 325-338 ◽  
Author(s):  
Roger M. Herriott
Keyword(s):  
Molecular Weight ◽  
Isoelectric Point ◽  
Specific Activity ◽  
Milk Clotting ◽  
Pepsin Inhibitor

A method has been described for the isolation and crystallization of swine pepsin inhibitor from swine pepsinogen. Solubility experiments and fractional recrystallization show no drift in specific activity. The reversible combination of pepsin with the inhibitor was found to obey the mass law. The inhibitor is quite specific, failing to act on other proteolytic and milk clotting enzymes. The inhibitor is destroyed by pepsin at pH 3.5. Chemical and physical studies indicate that the inhibitor is a polypeptide of approximately 5,000 molecular weight with an isoelectric point at pH 3.7. It contains arginine, tyrosine, but no tryptophane and has basic groups in its structure.

Isolation of the γc-globulin of bovine cerebrospinal fluid

1968 ◽  
Vol 46 (3) ◽  
pp. 273-276
Author(s):  
Catherine F. C. MacPherson ◽  
François Feldmuller
Keyword(s):  
Molecular Weight ◽  
Cerebrospinal Fluid ◽  
Gel Filtration ◽  
Globulin Fraction ◽  
Elution Volume ◽  
Immunological Activity ◽  
Immunochemical Method ◽  
Milk Whey ◽  
Preliminary Filtration ◽  
Gel Diffusion

The γc-globulin of bovine cerebrospinal fluid (CSF) was isolated in immunologically pure form from the globulin fraction of CSF or milk whey by gel filtration on Sephadex G-75. The globulin fraction was obtained by precipitation with sodium or ammonium sulfate rather than by DEAE chromatography because salt precipitation resulted in greater yields of the γc-globulin. When the protein was isolated from bovine colostral whey, about 90% of the immunoglobulin G (IgG) globulin was removed by a preliminary filtration of the whey on Sephadex G-75. The fraction containing the γc-globulin admixed with IgG globulin was then re-chromatographed on Sephadex G-75 to remove the IgG globulin. On gel filtration through a column of Sephadex G-75 that had been calibrated with proteins of known molecular weight, the elution volume of the γc-globulin corresponded to a molecular weight of 30 000. Different lots of γc-globulin that contained no detectable impurities by gel-diffusion tests were compared on a weight basis by a quantitative immunochemical method, and were found to have equal immunological activity.

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