Deterministic pressure-induced dissociation of vicilin, the 7S storage globulin from pea seeds: effects of pH and cosolvents on oligomer stability.

Abstract

A thermodynamic characterization of subunit association of vicilin, a storage protein from pea seeds, was performed using a combination of hydrostatic pressure and fluorescence spectroscopy. Application of pressure up to 2.4 kbar caused dissociation of vicilin subunits, as revealed by (1) size-exclusion PPLC of pressurized samples, (2) fluorescence anisotropy measurements of a dansyl-vicilin conjugate under pressure, and (3) quenching of the intrinsic fluorescence of vicilin. Pressure dissociation data were well described by a model for dissociation of a trimer. This enabled calculation of the standard molar volume change of association (delta Vo) and the equilibrium dissociation constant at atmospheric pressure (Ko); at pH 10 these were found to be delta Vo = 146 mL/mol and Ko = 2.2 x 10(-15) M2, respectively, corresponding to C1/2 (the concentration of protein at 50% dissociation at atmospheric pressure) = 18 nM and a stabilization free energy of -19.6 kcal/mol for the oligomer. Vicilin exhibited an anomalously low dependence on protein concentration for pressure dissociation. This appeared related to conformational changes in the dissociated subunits, which caused a loss of ca. 5 kcal/mol in the free energy of association and to structural/energetic heterogeneity in the population of oligomers. Pressure dissociation was markedly pH-dependent, with a stabilization free energy loss of 3.4 kcal/mol upon raising pH from 9 to 10. Circular dichroism and intrinsic fluorescence lifetime measurements at atmospheric pressure showed that the structure of vicilin was largely unaffected by pH in the range investigated. These results suggest that the effect of pH may involve deprotonation of lysine residues participating in salt bridges between vicilin subunits. Pressure dissociation of vicilin was significantly inhibited by addition of salts (NaCl, KCl, LiCl) or glycerol. Dissociation curves obtained in the presence of salts enabled calculation of the free energies of stabilization (ranging from ca. -1.2 to -2.4 kcal/mol) of the vicilin oligomers by these cosolvents. The similar effects of salts or glycerol suggest a common mechanism of stabilization of the oligomer involving exclusion of the cosolvents from the protein interface and preferential hydration of the protein.