Effects of types of sugar on the stabilization of protein in the dried state

The effects of various sugars on the structural stabilization of protein during freeze-drying were investigated. The degree of native structure of protein that was freeze-dried and rehumidified at constant relative humidities (RHs) was evaluated by measurement of the α-helix content by Fourier-transform infrared spectroscopy. Bovine serum albumin (BSA) and several types of sugars, including sucrose, trehalose, and dextrans, were used as a model protein and sugars, respectively. The glass transition temperature, T_g, for the amorphous sugar samples was measured by differential scanning calorimetry (DSC) to characterize the structural stability of sugars. The dependence of the α-helix content (C_α-helix) of BSA on the sugar content (C_sugar) could, in most cases, be represented by a Langmuir-type equation: C_α-helix =K x (C_α-helix^max -C_α-helix⁰) x C_sugar/(1 + K x c_sugar)+ C_α-helix⁰, where K is a constant, indicating the ability of amorphous sugar matrix to embed protein, and C_α-helix⁰ and C_α-helix^max indicate the α-helix content in the absence of sugar and saturating levels of sugar, respectively. The preservation effects of the sugars could be characterized by K and C_α-helix^max. Both K and C_α-helix^max values tended to be higher with decreasing T_g values for the amorphous sugar, probably because an amorphous sugar matrix with lower T_g values is structurally more flexible. The rehumidification of protein that was freeze-dried in the presence of sugar induced the refolding of protein structure, whereas the protein dried alone did not show any recovery of its native structure.