The aggregation of tris(4-glycosyloxyphenylazo)phloroglucinol compounds (Yariv antigens) where glyco = β-D-gluco, β-D-manno, β-D-galacto and α- D-galacto has been investigated by equilibrium sedimentation, the photoelectric scanning absorption optical system being utilized. The compounds have been shown to undergo a strong self-association in water and an analysis procedure employing Laplace transforms indicated a very wide distribution of species in the ultracentrifuge cell at equilibrium. To determine the type of association the concentration of monomer as a function of total weight concentration was calculated by using the recently developed Ω function. When the concentration of monomer is very low, as in the present case, the analysis is difficult because very slight curvature in the Ω-function plot has a large effect on the extrapolation to zero concentration. ��� The results are best described by an isodesmic indefinite self- association. The β-glucoside Yariv antigen was the most extensively investigated and the most highly purified sample was shown to have an association constant of 2.5 x 107 l. mol-1. The order of the association constants of the Yariv compounds was found to be β-glucoside > β- galactoside > α-galactoside > β-mannoside. For the β-glucoside compound an increase in temperature favours species of lower molecular weight, and from the enthalpy change a positive entropy change for the association is indicated. The compounds are disaggregated by concentrated solutions of urea and guanidine hydrochloride. Spectral measurements on the β-glucoside compound indicate disaggregation by sodium dodecyl sulfate above the critical micelle concentration.
Physicochemical studies are reported for conglutin �, the minor globulin isolated from seeds of L. angustifolius cv. Uniwhite. Isoelectric focusing of the native protein in polyacrylamide gel slabs resolved major and minor broad bands near pH 8.0 and 7.8 respectively. Following reduction of disulfide bonds with β-mercaptoethanol in 8 M urea, the smaller polypeptide chain of known sequence focused near pH 6.9 while the larger chain focused near pH 8.0. Sedimentation equilibrium studies showed that the major component in aqueous buffers at neutral pH is a hexamer of molecular weight 280 000 which dissociates to the monomer of molecular weight 47 000 at pH 4.8. The sequence molecular weight of the small subunit polypeptide is 16 517 [Elleman, T.C. (1977). Aust. J. Biol. Sci. 30, 33-45]. The molecular weights determined for the larger chain by sedimentation equilibrium or column chromatography in 6 M guanidine hydrochloride, and by dodecyl sulfate-polyacrylamide gel electrophoresis, were in the range 28 000-30 000. Optical rotatory dispersion and circular dichroism measurements have been used to establish the approximate proportions of α-helix (15%), β-structure (35%), β-turns (18%) and unordered regions (32%) in the native protein. The denaturation curve for guanidine hydrochloride and the proportions of α-helix (50%), β-turns (18%) and unordered regions (32%) in 80 % trifluoroethanol have been determined.
There have been many physicochemical studies of legumin, one of the major storage globulins isolated from pea seed. The more recent literature values for the molecular weight of this protein are in the range 390 000-420 000. These results are not consistent with the subunit molecular weight of legumin determined by dodecyl sulfate-polyacrylamide gel electrophoresis, if a hexameric model is assumed. We have measured the molecular weight of a highly purified sample of Pisum legumin by meniscus depletion sedimentation equilibrium and have found a value of 350 000 � 10 000. Since the oligomeric protein is homogeneous with respect to molecular weight, the heterogeneity reported for the subunit polypeptides, using various conditions of electrophoresis, presumably reflect differences in charge and amino acid composition. The molecular weight of legumin is significantly greater than the value of 325 000 found for cucurbitin, the equivalent crystalline protein isolated from pumpkin seed.
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