Apohorseradish peroxidase unfolding and refolding: Intrinsic tryptophan fluorescence studies

The unfolding and refolding of apohorseradish peroxidase, as a function of guanidinium chloride concentration, were monitored by the intrinsic fluores cence intensity, polarization, and lifetime of the single tryptophan residu e. The unfolding was reversible end characterized by at least three distinc t stages-the intensity and lifetime data, for example, were both characteri zed by an initial increase followed by a decrease and then a plateau region . The lifetime data, in the absence and presence of guanidinium chloride, w ere heterogeneous and fit best to a model consisting of a major Gaussian di stribution component and a minor, short discrete component. The observed in crease in intensity in the initial stage of the unfolding process is attrib uted to the conversion of this short component into the longer, distributed component as the guanidinium chloride concentration increases. Our results clarify and amplify previous studies on the unfolding of apohorseradish pe roxidase by guanidinium chloride.