A high affinity nonprecipitating anti-2,4-dinitrophenyl (DNP) IgG antibody constitutes a major fraction of circulating anti-DNP antibodies at the decline of the anti-DNP antibody response in pigs. The purified nonprecipitating antibody forms exclusively soluble complexes with DNP-PSA (pig serum albumin) or DNP-PGG (pig IgG). The binding sites are accessible to large ligands; one antibody molecule can combine with two molecules of DNP-PSA simultaneoulsy. The formation of complexes with DNP-PSA was studied in various media. No precipitate was formed in saline, in 0.9 M or 0.05 M sodium chloride, in 0.5 M potassium thiocyanate and in several nonionic detergent solutions (0.5% and 1%). Normal pig or guinea pig sera were found to contain a factor, removable by an antigen-antibody precipitate that caused a partial precipitation of the complexes. A mild alteration of the integrity of the antibody molecule by cleavage of interchain disulfide bonds did not affect the nonprecipitating character of the antibody. In media containing hydrophilic polymers, i. e. various dextrans, polyethylene glycols or a soluble polyamide (poly-a,P-[N(2-hydroxyethyl)-~L-aspartamide]), a functional conversion occurred; the antibody behaved like a precipitin. The precipitate with DNP-PSA could not be resolubilized upon removal of the polymer by washing. The magnitude of the functional conversion depended on the concentration of the polymer and on its molecular weight, larger molecules being more effective. The effect of the hydrophilic polymers could be potentiated by replacing water in the solvent partly by deuterium oxide. The findings are compatible with a notion, that in polymer-containing media the nonprecipitating antibody acquires a conformation functionally equivalent to the conformation of a precipitating antibody and that the altered conformation is stabilized by the lattice of the precipitate with DNP-PSA.
Diftalone showed a distinct inhibition effect on the collagen formation in sponge granuloma as well as in chick embryos articular cartilage. The inhibitory effect was expressed even in corneal and articular cartilage glycosaminoglycans (GAG) sulphation and GAG formation in granulation tissue. According to our previous results diftalone showed a similar inhibition of collagen and GAG biosynthesis as other effective antirheumatic drugs.
Pig antibodies to the dinitrophenyl group and fragments derived from them by limited proteolysis were studied by temperature-perturbation and solvcnt-perturbation spectroscopy with particular attention to differences between the number of perturbed chromophores in free antibodies and in antibody-hapten complexes.The position of the maxima in the difference spectra show that solvent-perturbed chromophores are exposed to water, but thermally perturbed chromophores are located in a microenvironment the polarity of which corresponds to 25 -SO % ethylenc glycol. A significant fraction of tyrosine residues (65 -90 z) and tryptophan residues (20-45 "/;;) is perturbed by temperature. Much lower fractions, i.e. [35][36][37][38][39][40][41][42][43][44][45] of tyrosine residues and less than 15 of tryptophan residucs, are perturbed by 20 "/, glycerol. The numbers of perturbed chromophorcs in fragments constituting the molecule are lower than or equal to the numbers in the original molecule.The effect of hapten binding is significant only with one of the antibody types, the prccipitating antibody. The number of thermally perturbed tyrosine residues is by about 17% lower in the liganded antibody. The absence of an analogous effect in the Fab fragment suggests that the fine conformational mechanism of signal transfer from the binding sites operates only in intact antibody molecules.
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