We report a systematic investigation of the effects of five carbohydrates (fructose, sucrose, trehalose, maltotriose, and dextran), freeze-drying, and subsequent heat treatment (up to 8 days at 80 degrees C) on the structure and enzymatic activity of alpha-chymotrypsin and catalase. Importantly, we investigate high carbohydrate-to-protein mass ratio conditions (20: 1 by mass) so as to minimize protein-protein interactions (e. g., protein aggregation). The activity of alpha-chymotrypsin is almost quantitatively recovered (greater than 80% of the initial activity) after lyophilization with or without added carbohydrates, and its conformation in the dried solid state as well as in an aqueous solution (after rehydration of lyophilized samples) is unchanged by the presence of carbohydrates. In contrast, catalase lyophilized without carbohydrates loses almost half of its activity upon reconstitution and shows significant structural changes in the dried state. The loss of activity and the lyophilization-induced structural alteration for catalase are appreciably reduced by addition of the lower molecular weight carbohydrates (fructose, sucrose, and trehalose). For protein preservation at elevated temperatures, the addition of carbohydrates at high carbohydrate-to-protein ratios (20: 1 by mass) creates a glassy matrix dilute with respect to the protein, effectively minimizing intermolecular protein-protein interactions that could lead to protein aggregation in the solid state. It is also shown that the Maillard reaction between carbohydrates and proteins, which causes a significant loss of enzymatic activity, is successfully suppressed in glassy systems with less than 1% (w/w) water content. More significantly, a quantitative comparison is established for the first time between activity loss and the extent to which the Maillard reaction proceeds for dry glassy systems (as measured spectroscopically). Trehalose was found to be the most effective protector against prolonged heating: approximately 90% of alpha-chymotrypsin activity and 30% catalase activity are recovered upon reconstitution following 8 days at 80 degrees C in a trehalose matrix.