Influence of native disulfide bonds of globular proteins on their retention behavior on reverse phase supports

The reduction of the disulfide bonds of globular proteins, for example, those of lysozyme or ribonuclease-A, results in an increase in the hydrodynamic volume of the polypeptide chain. This is reflected in an earlier elution of the reduced protein on gel filtration compared to that of the native disulfide-bonded form. The reduction of the four disulfide bonds of ribonuclease-A increased its retention time on reverse phase support, suggesting an increase in the "apparent hydrophobicity" of the protein molecule on reduction. Performic acid-oxidized ribonuclease-A eluted ahead of native disulfide-bonded ribonuclease on RP HPLC, suggesting a decrease in the hydrophobicity of the molecule. However, the hydrodynamic volume of performic acid-oxidized ribonuclease-A is similar to that of reduced protein as reflected in its gel filtration behavior. Thus, the increased retention of the reduced protein compared to that of native disulfide-bonded protein is not related to the increased hydrodynamic volume, and is a reflection of the stronger interaction of reduced protein with the reverse phase support. Reoxidation of the reduced ribonuclease-A regenerated the original chromatographic behavior of the protein on the reverse phase support. Similar results were also obtained with hen egg white lysozyme. The results of the present study are interpreted as indicating that the native disulfide bonds of a globular protein restrict the exposure of the hydrophobic amino acid residues of the polypeptide chain with a consequent lower retention on the reverse phase support compared to its reduced form.