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Unfolding and refolding of the constant fragment of the immunoglobulin light chain
Authors:Yuji Goto  Kozo Hamaguchi
Institution:Department of Biology, Faculty of Science Osaka University, Toyonaka, Osaka 560, Japan
Abstract:The kinetics of reversible unfolding and refolding by guanidine hydrochloride of the constant fragment of the immunoglobulin light chain are described. The kinetic measurements were made at pH 7.5 and 25 °C using tryptophyl fluorescence and farultraviolet circular dichroism.The kinetics of unfolding of the constant fragment showed two phases in the conformational transition zone and a single phase above the transition zone. A double-jump experiment confirmed the presence of two forms of the unfolded molecule. These results were thoroughly explained on the basis of the three-species mechanism, U1
/></figure> U<sub>2</sub><figure class=/></figure> N, where U<sub>1</sub> and U<sub>2</sub> are the slow-folding and fast-folding species, respectively, of unfolded protein and N is native protein. The equilibrium constant for the process of U<sub>2</sub> to U<sub>1</sub> was estimated to be about 10 and was independent of the conditions of denaturation. These findings were consistent with the view that the U<sub>1</sub><figure class=/></figure> U<sub>2</sub> reaction is proline isomerization. The rates of interconversion between N and U<sub>2</sub> changed greatly with the concentration of guanidine hydrochloride. On the other hand, the refolding kinetics below the transition zone showed behavior unexpected from the three-species mechanism. Whereas the apparent rate constant of the slow phase of refolding was independent of the refolding conditions, its amplitude decreased markedly with the decrease in the final concentration of guanidine hydrochloride. On the basis of this and other results, formation of an intermediate during refolding was ascertained and the refolding kinetics were consistently explained in terms of a more general mechanism involving a kinetic intermediate probably containing non-native proline isomers. The intermediate seemed to have a folded conformation similar to native protein. Comparison of the refolding kinetics of the constant fragment with those of other domains of the immunoglobulin molecule suggested that Pro143 is responsible for the appearance of the slow phase.</td> </tr> <tr> <td align=
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