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Evidence for the Existence of an Unfolding Intermediate of Thyroglobulin during Denaturation by Guanidine Hydrochloride

Y.-K. Hong1, F.-G. Meng1, H. Tang3, and H.-M. Zhou1,2*

1Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China; fax: 8610-627-72245; E-mail: zhm-dbs@tsinghua.edu.cn

2Protein Science Laboratory of the Ministry of Education, School of Life Science and Engineering, Tsinghua University, Beijing 100084, China

3Department of Chemistry, Guizhou Institute for Nationalities, Guiyang 550025, China

* To whom correspondence should be addressed.

Received March 11, 2002; Revision received March 19, 2002
The unfolding of bovine thyroglobulin (Tg) in guanidine hydrochloride (GuHCl) solution was studied by following the fluorescence and circular dichroism. With increasing GuHCl concentrations, the emission maximum of the intrinsic fluorescence clearly red-shifted in two stages. At concentrations of GuHCl less than 1.2 M or more than 1.6 M, the red shift showed a cooperative manner. At concentrations of GuHCl between 1.2 and 1.6 M, an unfolding intermediate was observed. It was further characterized by the increased binding of the fluorescence probe 1-anilinonaphthalene-8-sulfonic acid (ANS). No significant changes of the secondary structure were indicated by CD spectra at the concentrations of GuHCl between 1.2 and 1.6 M. The conformation of this state has properties similar to those of a molten globule state which may exist in the folding pathway of the protein. Further changes in fluorescence properties occurred at concentrations of denaturant higher than 1.6 M with a significant red shift of the emission maximum from 340 to 347 nm and a marked decrease in ANS binding. This in vitro study gave a clue to understand the biochemical mechanism for the occurrence of aggregation and molecular chaperone binding during Tg maturation in vivo.
KEY WORDS: thyroglobulin, guanidine hydrochloride, unfolding, intermediate