PK Changes of Ionizable Reporter Groups as an Index of Conformational Changes in Proteins. A Study of Fluorescein-Labelled Ribonuclease A

Abstract

A chemical derivative of bovine pancreatic RNase A was prepared by reaction with fluorescein-isothiocyanate at pH 6. This derivative has a fluorescein group covalently attached to the .alpha.-amino group of the protein. The enzymic properties of the modified protein are similar to those of RNase A. The pK of the fluorescein group can be used as an index of protein conformation to monitor structural changes in the protein. In this work, the binding of a specific inhibitor (cytidine 2''-monophosphate) to RNase A, the isomerization process occurring in RNase A around pH 6, and the thermal unfolding of RNase A, were studied using the pK changes of the fluorescein group. The results obtained by this method are fully consistent with those obtained by other methods. Using ionizable reporter groups and their changes in pK to monitor conformational changes in proteins may be a sensitive tool both in equilibrium and kinetic studies.