pH dependence of the reversible and irreversible thermal denaturation of .gamma. interferons

Abstract

Heated at pH 6.0 and at 50.degree. C, human interferon .gamma. (HuIFN-.gamma.) is inactivated via the formation of insoluble aggregates. At pH 6.0, the aggregation rate increases with temperature from 40 to 65.degree. C. There is a temperature-dependent time lag to aggregate formation observed in the generation of light-scattering particles at pH 6.0, and this correlates with the fast phase observed in the kinetics of reversible thermal unfolding. In addition, the dependence of aggregation kinetics on temperature closely follows the reversible melting curve. These observations suggest that at pH 6.0 irreversible thermal denaturation and aggregation depend on partial or complete unfolding of the molecule. At pH 5.0, also at 50.degree. C, the molecule is stable to irreversible aggregation. In reversible unfolding in 0.25 M guanidine hydrochloride, the Tm for HuIFN-.gamma. increases from 30.5.degree. C at pH 4.75 to 41.8.degree. C at pH 6.25, in analogy to the behavior of other globular proteins. These observations suggest that the relative instability of HuIFN-.gamma. to irreversible denaturation via aggregation at pH 6.0 compared to pH 5.0 is not due to an increased stability toward unfolding at the lower pH. Alternatively, stability at pH 5.0 must be due either to the improved solution properties of the unfolded state or to the improved solubility/decreased kinetic lifetime of an unfolding intermediate. Aggregation of HuIFN-.gamma. at 50.degree. C is half-maximal at pH 5.7, suggesting that protonation of one or both of the histidine residues may be involved in this stabilization. This hypothesis is supported by chemical modification experiments in which reaction with the histidine-specific reagent ruthenium pentaamine eliminates aggregation of the thermally unfolded molecule at pH 6.0. Histidine involvement may also explain our finding that rabbit interferon .gamma. and bovine interferon .gamma. do not aggregate in the pH range of 5.0-6.0 at temperatures close to their Tms.