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Relationship between conformational stability and lyophilization‐induced structural changes in chymotrypsin
Author(s) -
Carrasquillo Karen G.,
Sanchez Cheryll,
Griebenow Kai
Publication year - 2000
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1042/ba19990087
Subject(s) - chemistry , aqueous solution , freeze drying , protein secondary structure , protein structure , conformational change , crystallography , trehalose , native state , structural stability , denaturation (fissile materials) , circular dichroism , chromatography , biophysics , stereochemistry , biochemistry , organic chemistry , nuclear chemistry , structural engineering , biology , engineering
The relationship between protein conformational stability in aqueous solution and the magnitude of lyophilization‐induced structural changes was investigated employing α‐ and γ‐chymotrypsin. As a measure of the conformational stability the melting temperature T m was determined in distilled water at various pH values. The proteins were then lyophilized from those pH values where the conformational stability was maximum (pH 4.5) and minimum (pH 7.8). Protein secondary structure was quantitatively determined utilizing Fourier‐transform infrared spectroscopy employing two regions sensitive to protein structure, the amide‐I (1600–1700 cm −1 ) and amide‐III (1215–1335 cm −1 ). Lyophilization induced significant structural alterations in both proteins, characterized by a slight decrease in the α‐helix and a significant increase in the β‐sheet content. However, regardless of the pH from which the proteins were lyophilized, the secondary structures in the solid state were indistinguishable. This result shows that there is no relationship between the conformational stability in aqueous solution and the magnitude of lyophilization‐induced structural changes. We also investigated whether lyoprotectants could minimize lyophilization‐induced structural changes 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‐lyophilization experiments from various pH values. The results obtained exclude any contribution from increased protein conformational stability caused by the additive in aqueous solution to the beneficial structural preservation upon lyophilization. This can be understood because the dehydration and not the freezing process, as shown in an air‐drying experiment, mainly causes protein structural alterations.