Sedimentation Equilibrium of Low‐molecularweight Associating Solutes

Holde, Kensal E. van; Rossetti, Giulia; Dyson, Robert D.

doi:10.1111/j.1749-6632.1969.tb14044.x

Cited by 49 publications

(21 citation statements)

References 16 publications

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“…The discrete calculation of van Holde et al (14) was also applied to the weight-average molecular weight data. Table 1A shows the stoichiometries, which yielded positive equilibrium constants when tested to hexamers of the units weighing 292 X 103 g/mol.…”

Section: Resultsmentioning

confidence: 99%

“…Plates were read on an automatic microdensitometer linked to a PDP-12 computer (13). For analysis of selfassociating stoichiometries, the procedures and computer programs discussed by van Holde et al (14) and Teller- (15) were used. For the analysis of complex formation between dissimilar molecules, the procedure and computer program of Behnke et al (12) were utilized.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

The Molecular Basis of Self-Association of Antibodies to IgG (Rheumatoid Factors) in Rheumatoid Arthritis

Pope

Teller

Mannik

1974

Proc. Natl. Acad. Sci. U.S.A.

122

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The serum and synovial fluid of many patients with rheumatoid arthritis contain immune complexes composed of immunoglobulin G (IgG). In this study such complexes from one patient are shown to be formed by self-association of IgG-antibodies to IgG (IgG-rheumatoid factors), so that each molecule serves as an antibody as well as an antigen. All F(ab')s and Fab' fragments derived from these complexes have antibody binding sites for normal IgG. Due to a high association constant in the formation of a cyclic complex by these antibodies, normal IgG is excluded as an antigen. These studies serve as a model for further elucidation The isolated complexes or the F(ab')2 fragments derived from these complexes were trace-labeled with 125I at 1-2 mol of iodine per mol of protein (7). Radioactivity was measured by an automatic well-type gamma counter.The immunological purity of complexes was ascertained by double diffusion in 2% agarose in borate buffer, using monospecific antisera to y, a, yt, K, and X chains of immunoglobulins (8) and an antiserum to human serum. A search for idiotypic antigenic determinants (9) was made by hyperimmunizing six rabbits with the purified complexes in complete Freund's adjuvant. Antisera were obtained 3 and 10 weeks after the last immunization. The presence of precipitating idiotypic antibodies was examined by double diffusion in agarose.Several methods were employed to examine antibody activity in complexes or their fragments. For these systems pooled normal human IgG was obtained from Cohn Fraction II (Schwarz/Mann, Orangeburg, N.Y.) by ion-exchange chromatography on DEAE. The material eluted with 0.01 M phosphate buffer, pH 8.0, was used for obtaining monomeric IgG by gel filtration on Sephadex G-200. Heat-aggregated human IgG was prepared from Cohn Fraction II, by the method of Christian; only fractions SS, and SS2 were employed (10).Quantitative assays for binding of complexes or their F(ab')2 fragments to normal 6.6S IgG or aggregated IgG were performed. Radiolabeled complexes or their fragments were added to IgG or aggregated IgG, incubated at 230 for 1 hr and at 40 for 1-2 hr. The interaction of these reactants was examined by distribution of radioactivity after zone ultracentrifugation. Previously described procedures were employed for zone ultracentrifugation (11). Either borate (0.2 M sodium borate, 0.15 M NaCl, pH 8.0) or acetate (0.05 M sodium acetate, 0.15 M NaCl, pH 3.5) buffers were used to build linear sucrose gradients.Analytical Ultracentrifugation. Analytical ultracentrifugation studies were performed with a Beckman-Spinco model E analytical ultracentrifuge equipped with both the schlieren and

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

The Molecular Basis of Self-Association of Antibodies to IgG (Rheumatoid Factors) in Rheumatoid Arthritis

Pope

Teller

Mannik

1974

Proc. Natl. Acad. Sci. U.S.A.

122

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“…Editing of the raw data, obtained from the plate-reading program as well as the calculation of the point by point number (M,), weight (M,), and z (M,) average molecular weights were accomplished by previously described techniques (13,14). Analysis of the high-speed equilibrium data for self-association stoichiometry and calculations of the association constants were performed with computer programs based on the methods of van Holde et a1 (15).…”

Section: Methodsmentioning

confidence: 99%

Self‐associating igg rheumatoid factors in mrl/1 autoimmune mice

Nardella

Teller

Izui

et al. 1984

Arthritis & Rheumatism

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Previous work has shown that the intermediate complexes isolated from the plasma of patients with rheumatoid arthritis are composed of self-associating IgG rheumatoid factors. Mice of the MRLh strain develop spontaneous autoimmune disease with arthritis that is pathologically similar to human rheumatoid arthritis. Also, the sera of MRL/I mice contain autoantibodies to nuclear antigens as well as IgM and IgG rheumatoid factors. The present studies were done to determine if the IgG rheumatoid factors isolated from these mice undergo self-association. MRL/I mouse sera were categorized into groups A and B based on serumserum precipitin interactions. Thirteen of 13 MRWl mice sera examined contained intermediate complexes sedimenting between the 6.6s and 19s components of normal serum by sedimentation velocity ultracentrifu- gation. There were no differences in the level of intermediate complexes between groups A and B. IgG rheumatoid factors were isolated from the sera of 9 other mice. Upon sedimentation equilibrium ultracentrifugation, these rheumatoid factors underwent concentration-dependent self-association similar to that described for human self-associating IgG rheumatoid factors, although the precise stoichiometry of self-association could not be determined. The IgG rheumatoid factors from group B had higher energies of self-interaction than those from group A. These studies provide additional evidence that MRL/l mice may be the best available animal model for the study of human rheumatoid arthritis.

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“…While we have demonstrated that reasonable precision can be attained at low concentrations (fringe displacements) providing special care is taken in the experimental procedures and measurements, the overall precision is not high. This can be a serious disadvantage when one is trying to decide between definite and indefinite association for a given system since very high precision is then required (see also Van Holde, Rossetti, and Dyson 1969). A second serious problem may arise from the assumption made in treating the results that v is the same for all forms of the protein present.…”

Section: Discussionmentioning

confidence: 99%

On the Dissociation of Bovine ,b-Lactoglobulins A, B, and C Near Ph 7

McKenzie¹,

Sawyer²

1972

Aust. Jnl. Of Bio. Sci.

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Sedimentation-equilibrium studies are made of the moleoular weights of bovine ,B.laotoglobulins A, B, and C at pH 7·5. The order of dissooiation of dimer to monomer is A> B ~ C. The dissooiation oonstant (Kd) for A and B is 0·6 X 10-4 and 0·08x 10-4 mole 1-1 at 20°C, respeotively. The oonditions ohosen for these measurements are based on sedimentation velooity studies in the pH range 6-9. There is no ohange in sedimentation velooity behaviour following different times of standing at 20°C for pH < 7·5. The sedimentation patterns exhibit a single peak with some trailing on the solvent side. At low oonoentrations the plot of weight average sedimentation coefficient (,~) versus concentration (0) is in aocord with that of a rapidly dissociating system of monomer-dimer type. There are time-dependent aggregations above pH 8. Effects of changes in ionic strength and addition of methanol are considered.Using several values of K d , .9 versus 0 ourves are oaloulated by the method of Gilbert (1963) and oompared with the present experimental ourves and also those of Zimmerman, Barlow, and Klotz (1970). The agreement between theory and experiment is only moderate.

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Sedimentation Equilibrium of Low‐molecularweight Associating Solutes

Cited by 49 publications

References 16 publications

The Molecular Basis of Self-Association of Antibodies to IgG (Rheumatoid Factors) in Rheumatoid Arthritis

The Molecular Basis of Self-Association of Antibodies to IgG (Rheumatoid Factors) in Rheumatoid Arthritis

Self‐associating igg rheumatoid factors in mrl/1 autoimmune mice

On the Dissociation of Bovine ,b-Lactoglobulins A, B, and C Near Ph 7

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