Relationship between conformational stability and lyophilization-induced structural changes in chymotrypsin

The relationship between protein conformational stability in aqueous soluti on and the magnitude of lyophilization-induced structural changes was inves tigated employing alpha- and gamma-chymotrypsin. As a measure of the confor mational stability the melting temperature T-m was determined in distilled water at various pH values. The proteins were then lyophilized from those p H values where the conformational stability was maximum (pH 4.5) and minimu m (pH 7.8), Protein secondary structure was quantitatively determined utili zing Fourier-transform infrared spectroscopy employing two regions sensitiv e to protein structure, the amide-l (1600-1700 cm(-1)) and amide-III (1215- 1335cm(-1)). Lyophilization induced significant structural alterations in b oth proteins, characterized by a slight decrease in the alpha-helix and a s ignificant increase in the beta-sheet content. However, regardless of the p H from which the proteins were lyophilized, the secondary structures in the solid state were indistinguishable. This result shows that there is no rel ationship between the conformational stability in aqueous solution and the magnitude of lyophilization-induced structural changes. We also investigate d whether lyoprotectants could minimize lyophilization-induced structural c hanges by increasing protein conformational stability in aqueous solution. After having identified trehalose as being efficient in largely preventing lyophilization-induced structural alterations, we conducted co-lyophilizati on experiments from various pH values. The results obtained exclude any con tribution from increased protein conformational stability caused by the add itive in aqueous solution to the beneficial structural preservation upon ly ophilization. This can be understood because the dehydration and not the fr eezing process, as shown in an air-drying experiment, mainly causes protein structural alterations.