Effect of freezing and thawing rates on denaturation of proteins in aqueous solutions

Effect of freezing and thawing rates on denaturation of proteins in aqueous solutions
Published Online: 24 Mar 2003
Received: 11 July 2002; Accepted: 7 November 2002
Enhong Cao 1, Yahuei Chen 1, Zhanfeng Cui 1 *, Peter R. Foster 2
Biotechnology and Bioengineering
Volume 82, Issue 6 , Pages 684 - 690
Wiley InterScience
1Department of Engineering Science, University of Oxford, Parks Road, Oxford OX1 3PJ, United Kingdom
2Protein Fractionation Centre, Scottish National Blood Transfusion Service, Edinburgh, United Kingdom

email: Zhanfeng Cui (zhanfeng.cui@eng.ox.ac.uk)
*Correspondence to Zhanfeng Cui, Department of Engineering Science, University of Oxford, Parks Road, Oxford OX1 3PJ, United Kingdom
Funded by:
Biotechnology and Biological Science Research Council (BBSRC) of the U.K.
Keywords
protein freezing ? freeze denaturation ? freezing rate ? thawing rate ? buffer type ? lactate dehydrogenase ? cryomicroscope
Abstract
The freeze denaturation of model proteins, LDH, ADH, and catalase, was investigated in absence of cryoprotectants using a microcryostage under well-controlled freezing and thawing rates. Most of the experimental data were obtained from a study using a dilute solution with an enzyme concentration of 0.025 g/l. The dependence of activity recovery of proteins on the freezing and thawing rates showed a reciprocal and independent effect, that is, slow freezing (at a freezing rate about 1?C/min) and fast thawing (at a thawing rate >10?C/min) produced higher activity recovery, whereas fast freezing with slow thawing resulted in more severe damage to proteins. With minimizing the freezing concentration and pH change of buffer solution by using a potassium phosphate buffer, this phenomenon could be ascribed to surface-induced denaturation during freezing and thawing process. Upon the fast freezing (e.g., when the freezing rate >20?C/min), small ice crystals and a relatively large surface area of ice-liquid interface are formed, which increases the exposure of protein molecules to the ice-liquid interface and hence increases the damage to the proteins. During thawing, additional damage to proteins is caused by recrystallization process. Recrystallization exerts additional interfacial tension or shear on the entrapped proteins and hence causes additional damage to the latter. When buffer solutes participated during freezing, the activity recovery of proteins after freezing and thawing decreased due to the change of buffer solution pH during freezing. However, the patterns of the dependence on freezing and thawing rates of activity recovery did not change except for that at extreme low freezing rates (<0.5?C/min). The results exhibited that the freezing damage of protein in aqueous solutions could be reduced by changing the buffer type and composition and by optimizing the freezing-thawing protocol. ? 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 82: 684-690, 2003.
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