Detection of intermediates in protein folding of carbonic anhydrase with fluorescence emission and polarization.

The three-dimensional structure of carbonic anhydrase is a result of specific folding of the protein chain to form a compact, globular molecule. Fluorescence measurements on the nature of the rate-limiting steps in folding from the random coil to the native structure show that each step involves an actual folding reaction of the protein chain. Emission intensity and polarization of the intrinsic fluorescence due to tryptophan residues reach a maximum during the early period of the folding process. The changes occur in at least three kinetic phases (tau1 less than 3 S, tau2 = 1 min, tau3 = 10 min, 1 M guanidinium chloride, 2 M NaC1, pH 7, 20 degrees C). None of these phases are explained by configurational changes in the fully unfolded chain. The results are consistent with a kinetic scheme that involves stepwise acquisition of the specific folded structure of the native enzyme.