For use in protein-folding studies, a rapid procedure for the preparation of octa-S-sulforibonuclease A (SO3-RNase A) with 2-nitro-5-(sulfothio)benzoate is described. The modification is specific for thiols and disulfide bonds. The modified protein was characterized and found to be enzymatically inactive and predominantly conformationally disordered. In the absence of thiols, the modified sulfhydryl groups were found to be stable over the pH range of 2-9. However, when the modified protein is incubated at neutral to slightly alkaline conditions for prolonged periods of time or at elevated temperatures, it undergoes a further (irreversible) modification that decreases its net charge at pH 8.0. Evidence is presented that demonstrates that this additional modification is due to the specific deamidation of asparagine-67. When incubated with an excess of reduced and oxidized glutathiones for 24 h at pH 8.2 and 25 degrees C, the reversible sulfo blocking group was removed, and essentially quantitative (94%) native enzymatic activity was regenerated from both SO3-RNase A and its deamidated derivative (SO3-RNase B). Although the two fully active refolded species differ in their elution behavior on ion-exchange chromatography, they are indistinguishable by many other methods. The significance of this finding for studies of the folding of RNase A is discussed.