Research Articles
Effects of Types of Sugar on the Stabilization of Protein in the Dried State

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ABSTRACT

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, Tg, 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 (Cα-helix) of BSA on the sugar content (csugar) could, in most cases, be represented by a Langmuir-type equation: Cα-helix = K × (Cα-helixmax − Cα-helix0) × csugar/(1 + K × csugar) + Cα-helix0, where K is a constant, indicating the ability of amorphous sugar matrix to embed protein, and Cα-helix0 and Cα-helixmax 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 Cα-helixmax. Both K and Cα-helixmax values tended to be higher with decreasing Tg values for the amorphous sugar, probably because an amorphous sugar matrix with lower Tg 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.

Section snippets

INTRODUCTION

Preserving the native conformation of protein during freeze-drying would lead to a greater stability against denaturation during storage.1., 2., 3., 4., 5. A number of investigators have reported that freeze-drying in the presence of sugar aids in preserving the native conformation of a protein.6., 7., 8., 9., 10., 11. When a protein solution is freeze-dried in the presence of sugars, amorphous matrices of sugars are formed, which could embed protein molecules. The embedding of protein

Materials

Glucose, sucrose, maltose, trehalose, and maltotriose were purchased from Wako Pure Chemical Industries Ltd., (Osaka, Japan), and two types of dextran from Leuconostoc sp., with average molecular masses of 1500 (dextran 1500) and 6000 (dextran 6000), were obtained from Fluka Chemie AG (Buchs, Switzerland). Bovine serum albumin (BSA, 3× crystallized) was obtained from Sigma Chemical Company (St. Louis, MO). P2O5, LiCl, CH3COOK, K2CO3, and KBr were purchased from Wako Pure Chemical Industries

Glass Transition Temperatures for Amorphous Sugars

The Tg values for various amorphous sugars, as well as those in literature, are listed in Table 1. The values listed under given conditions vary considerably, probably because of the different conditions for sample preparations and DSC measurements. Hence, the values obtained in this study were used as the index of the physical property of the amorphous matrix of sugar. The Tg value for the glucose alone sample could not be measured because the amorphous glucose collapsed during

DISCUSSION

In this paper, we determined the effects of amorphous sugar matrices on the preservation of BSA, a model protein, in the dried state. In particular, the effects of different types and amounts of sugar were investigated with respect to the change in α-helix content of BSA. The relationship between sugar content and α-helix content could be approximated reasonably well by the Langmuir-type equation shown by eq. 2, in which the K value indicates the ability of the sugar matrix to embed protein

CONCLUSIONS

The preservation ability of various sugars on freeze-dried BSA was investigated under various conditions using the change in the α-helix content of BSA as an index. The dependency of the α-helix content on sugar content could be approximated by a Langmuir-type equation. The preservation ability of sugars was characterized by the two parameters appearing in the equation, K and Cα-helixmax, which represent the abilities of sugars to embed protein molecules and the maximum preserved α-helix

ACKNOWLEDGEMENTS

This work was supported by a grant-in-aid for the Encouragement of Young Scientists, No. 10750548 and 12750664, from the Ministry of Education, Science, Sport, and Culture of Japan.

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