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J. Biol. Chem., Vol. 282, Issue 46, 33452-33458, November 16, 2007

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Effect of High Concentration of Inert Cosolutes on the Refolding of an Enzyme

CARBONIC ANHYDRASE B IN SUCROSE AND FICOLL 70^*

From the Laboratory of Biochemistry and Genetics, NIDDK, National Institutes of Health, Bethesda, Maryland 20892

The kinetics of refolding of carbonic anhydrase II following transfer from a buffer containing 5 M guanidinium chloride to a buffer containing 0.5 M guanidinium chloride were studied by measuring the time-dependent recovery of enzymatic activity. Experiments were carried out in buffer containing concentrations of two "inert" cosolutes, sucrose and Ficoll 70, a sucrose polymer, at concentrations up to 150 g/liter. Data analysis indicates that both cosolutes significantly accelerate the rate of refolding to native or compact near-native conformations, but decrease the fraction of catalytically active enzyme recovered in the limit of long time. According to the simplest model that fits the data, both cosolutes accelerate a competing side reaction yielding inactive compact species. Acceleration of the side reaction by Ficoll is significantly greater than that of sucrose at equal w/v concentrations.

Received for publication, June 22, 2007 , and in revised form, August 14, 2007.

^* This work was supported by the Intramural Program of NIDDK, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Tel.: 301-594-2195; Fax: 301-402-0240; E-mail: monterrosob{at}niddk.nih.gov.

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