ABSTRACT
Introduction ........................................................................................................ 543
Materials and Methods ..................................................................................... 544
Preparation of Freeze-Dried LDH Sample, Assay of the LDH
Activity, and DSC Measurement ......................................................... 544
Preparation of Freeze-Dried Lysozyme Sample and FT-IR Study..... 545
Results.................................................................................................................. 546
The Loss of LDH Activity Caused by Freeze-Drying and
Long-Term Storage................................................................................. 546
The Effect of Trehalose and Residual Water on the Degree of the
Hydration Structure of Freeze-Dried Lysozyme .............................. 547
Discussion ........................................................................................................... 548
Acknowledgments ............................................................................................. 549
References ........................................................................................................... 549
Freeze-drying has been commonly used in both the food and pharmaceutical
industries as a long-term stabilizing method for unstable proteins. However,
since proteins are damaged by numerous stresses involved in the freeze-
drying process, it is necessary to add an agent such as trehalose to protect the
proteins from these stresses (Crowe, 2002). The protein protection
mechanism of trehalose upon freeze-drying has been primarily explained
by two effects: a water-replacement effect in which amorphous trehalose
plays the role of the water substitution forming a hydrogen bond with the
protein (Carpenter and Crowe, 1989; Suzuki et al., 1998; Wolkers et al., 1998;
Allison et al., 1999; Crowe, 2002); and the glass-transition effect in which a
glassy matrix formed by amorphous trehalose controls the molecular
mobility of the protein (Suzuki et al., 1998; Wolkers et al., 1998; Allison et al.,
1999; Crowe, 2002). Although these concepts are commonly acknowledged
as the stabilizing mechanisms for proteins by amorphous trehalose, the
influence of the residual water on the stabilizing effect remains unclear. It is
well known that residual water not only behaves as a reactant in the
chemical reactions such as deamidation (Hsu et al., 1991), but also sharply
lowers the glass-transition temperature (T
) of glassy matrix by the
plasticizing effect (Crowe, 2002). These effects of residual water promote
the whole reactivity in the system and, therefore, the concept “the drier the
better” has been commonly accepted (Hsu et al., 1991). However, from the
viewpoint of maintaining protein structure, over-drying may not necessarily
be a suitable method, even in the case in which amorphous trehalose
functions to maintain the protein structure by water substitution. This is
thought to be because trehalose is larger than water and is unable fit into all
the hydration sites in the protein because of steric hindrance. Thus, residual
water may be able to fill the remaining hydration sites. In order to evaluate
the validity of this proposal, the following two experiments were carried out.
First, the enzymatic activity of lactate dehydrogenase (LDH) was investi-
gated in order to determine the influence of amorphous trehalose and
residual water on the loss of LDH activity caused by freeze-drying and long-
term storage. The T
