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J Phys Chem B. 2013 Apr 4;117(13):3554-9. doi: 10.1021/jp4006923. Epub 2013 Mar 25.

Compensating effects of urea and trimethylamine-N-oxide on the heteroassociation of α-chymotrypsin and soybean trypsin inhibitor.

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Section on Physical Biochemistry, Laboratory of Biochemistry and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, National institutes of Health, U.S. Department of Health and Human Services, Bethesda, Maryland 20892, USA.


An assay for the determination of the equilibrium constant for heteroassociation of α-chymotrypsin and soybean trypsin inhibitor via fluorescence depolarization is described. Results obtained at neutral pH in saline buffer were consistent with prior determinations via sedimentation equilibrium and static light scattering. The dependence of the association equilibrium constant upon the concentrations of urea and trimethylamine-N-oxide (TMAO) added individually and in mixtures was determined at several temperatures. It was found that subdenaturing concentrations of urea decrease the extent of heteroassociation and that added TMAO increases the extent of heteroassociation. The effects of both cosolutes in mixtures upon the equilibrium heteroassociation of α-chymotrypsin and soybean trypsin inhibitor appear to be additive. A thermodynamic analysis of the combined results is presented.

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