GdmCl-, urea-, and pH-induced unfolding pathways of bovine carbonic anhydrase II have been analyzed by using changes induced by different denaturing agents in intensity, anisotropy, life time, and parameter A value of intrinsic fluorescence as well as intensity and life time of ANS (ammonium salt of 8-anilinonaphthalene-1-sulfonic acid) fluorescence. The formation of several stable unfolding intermediates, some of which were not observed previously, has been established. This was further confirmed by representation of fluorescence data in terms of a "phase diagram", that is, I(lambda1) versus I(lambda2) dependence, where I(lambda1) and I(lambda2) are the fluorescence intensity values measured at wavelengths lambda(1) and lambda(2), respectively.

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About 30% of proteins require cofactors for their proper folding. The effects of cofactors on the folding reaction have been investigated with a-lactalbumin as a model protein and metal ions as cofactors. Metal ions accelerate the refolding of a-lactalbumin by lessening the energy barrier between the molten globule state and the transition state, mainly by decreasing the difference of entropy between the two states. These effects are linked to metal ion binding to the protein in the native state. Hence, relationships between the metal affinities for the intermediate states and those for the native state are observed. Some residual specificity for the calcium ion is still observed in the molten globule state, this specificity getting closer in the transition state to that of the native state. The comparison between kinetic and steady-state data in association with the F value method indicates the binding of the metal ions on the unfolded state of a-lactalbumin. Altogether, these results provide insight into cofactor effects on protein folding. They also suggest new possibilities to investigate the presence of residual native structures in the unfolded state of protein and the effects of such structures on the protein folding reaction and on protein stability. In the presence of cofactors, the stability of the native state is increased and the folding reaction is dramatically accelerated. The latter observation further implies that cofactors can bind to unfolded or partially folded states of proteins. Nevertheless, the cofactor effects on the folding reaction remain poorly understood. Better characterization of their effects on the structure and energetics of the intermediate folding states are necessary. Toward that purpose, we report here a study with bovine a-lactalbumin (BLA) as a protein model and metal ions as cofactors.BLA is a small a + b milk protein with a calcium ion (Ca Article published online ahead of print. Article and publication date are at