Abstract: The renaturation of denatured ribonuclease was studied by covalent bonding the glutathione/glutathione disulfide(GSH/GSSG) to the surface of stationary phase. The results showed that the glutathione bonding column had a typical characters of weak-cation exchange, under the mode of ion-exchange chromatography(IEC), the column had a better column efficiency and four kinds of standard proteins could be baseline separated. With the protein concentration of 5 mg/mL, flow rate of 0.2 mL/min and no GSH/GSSG in mobile phase, the bioactivity recovery of denatured ribonuclease could reach (39.5?3.8)% refolded by glutathione bonding column, which was almost zero by common IEC column. The glutathione bonding column had an evident promoter action to the right refolding of denatured proteins disulfide bonds. Moreover, the bioactivity recovery could reach (81.5?4.3)% by adding GSH/GSSG to sample collections directly. These results may be useful for the development of protein folding liquid chromatography(PFLC) and in reducing the cost of proteins folding.

Key words: Protein folding, Ribonuclease, Ion-exchange chromatography, Glutathione bonding column