The effect of adsorption onto aluminum salt adjuvants on the structure and stability of three model protein antigens was studied using fluorescence and Fourier transform infrared spectroscopies, as well as isothermal titration and differential scanning calorimetric techniques. Lysozyme was preferentially adsorbed to aluminum phosphate (Adju-Phos®), whereas ovalbumin and bovine serum albumin were better adsorbed to aluminum hydroxide (Alhydrogel®). A linearized Langmuir adsorption isotherm was used to obtain information regarding the binding interactions between proteins and adjuvants. Binding energetics and stoichiometry data obtained from isothermal titration calorimetry measurements were complex. Based on the spectroscopic and differential scanning calorimetry studies, the structure of all three proteins, when adsorbed to the surface of an aluminum salt, was altered in such a way as to render the proteins less thermally stable. Besides the pharmaceutical significance of this destabilization, we consider the possibility that this phenomenon may facilitate the presentation of antigens and thus contribute to the adjuvant activity of the aluminum salts.

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Agitation- and freeze-thawing-induced aggregation of recombinant human factor XIII (rFXIII) is due to interfacial adsorption and denaturation at the air-liquid and ice-liquid interfaces. The aggregation pathway proceeds through soluble aggregates to formation of insoluble aggregates regardless of the denaturing stimuli. A nonionic surfactant, polyoxyethylene sorbitan monolaurate (Tween 20), greatly reduces the rate of formation of insoluble aggregates as a function of surfactant concentration, thereby stabilizing native rFXIII. Maximum protection occurs at concentrations close to the critical micelle concentration (cmc), independent of initial protein concentration. To study the mechanistic aspects of the surfactant-induced stabilization, a series of spectroscopic studies were conducted. Electron paramagnetic resonance spectroscopy indicates that binding is not occurring between Tween 20 and either the native state or a folding intermediate state of rFXIII. Further, circular dichroism spectroscopy suggests that Tween 20 does not prevent the secondary structural changes induced upon guanidinium hydrochloride-induced unfolding. Taken together, these results imply that Tween 20 protects rFXIII against freeze-thawing- and agitation-induced aggregation primarily by competing with stress-induced soluble aggregates for interfaces, inhibiting subsequent transition to insoluble aggregates.

Osmolytes increase the thermodynamic conformational stability of proteins, shifting the equilibrium between native and denatured states to favor the native state. However, their effects on conformational equilibria within native-state ensembles of proteins remain controversial. We investigated the effects of sucrose, a model osmolyte, on conformational equilibria and fluctuations within the native-state ensembles of bovine pancreatic ribonuclease A and S and horse heart cytochrome c. In the presence of sucrose, the farand near-UV circular dichroism spectra of all three native proteins were slightly altered and indicated that the sugar shifted the native-state ensemble toward species with more ordered, compact conformations, without detectable changes in secondary structural contents. Thermodynamic stability of the proteins, as measured by guanidine HCl-induced unfolding, increased in proportion to sucrose concentration. Nativestate hydrogen exchange (HX) studies monitored by infrared spectroscopy showed that addition of 1 M sucrose reduced average HX rate constants at all degrees of exchange of the proteins, for which comparison could be made in the presence and absence of sucrose. Sucrose also increased the exchange-resistant core regions of the proteins. A coupling factor analysis relating the free energy of HX to the free energy of unfolding showed that sucrose had greater effects on large-scale than on small-scale fluctuations. These results indicate that the presence of sucrose shifts the conformational equilibria toward the most compact protein species within native-state ensembles, which can be explained by preferential exclusion of sucrose from the protein surface.