Circular dichroism studies on the effects of ethanol on the conformation of α1-acid glycoprotein, α1-antitrypsin, deoxyribonuclease, pepsinogen, soybean trypsin inhibitor and unfolded ribonucleases

Abstract

The effects of 25 to 75 volume-% ethanol on conformation of human serum α 1-acid glycoprotein, human serum α 1-antitrypsin, pancreatic deoxyribonuclease I, porcine pepsinogen, the “Kunitz” trypsin inhibitor from soybeans, and oxidized as well as reduced and S-carboxymethylated ribonucleases were tested by the circular dichroism (CD) probe. It was found that 25 volume-% ethanol had a slight effect, whereas 50–75 vol.-% alcohol significantly altered the conformation. The tertiary structure was perturbed and the polypeptide main chain was reorganized into new conformations of higher helix and β-structure contents than in the native state. Comparison of the various proteins showed that the degree of reorganization depended chiefly on the cross-linking of the main chain by disulfide bridges. While the unfolded ribonucleases were refolded by 25 vol.-% ethanol into ordered conformations, the native ribonuclease was resistant even to 50–66% alcohol. The conformation of deoxyribonuclease and α 1-antitrypsin was more sensitive to 25 vol.-% ethanol than the conformation of α 1-acid glycoprotein, pepsinogen, and soybean trypsin inhibitor. Almost complete restoration of the native conformation was achieved by diluting the alcohol-containing solutions with water or by dialysis against water or buffer solutions. However, the renaturation depended on the time of contact with alcohol and on the temperature at which the alcohol-containing solutions were kept.