Denaturation of Globular Proteins

The techniques of equilibrium dialysis and ultrafiltration were utilized to study the interaction of urea, thiourea, methylurea, tetramethylurea, acetamide, ethylene glycol, and dextrose with bovine plasma albumin in water. It was found that the addition of anhydrous protein to water containing a nondenaturant (dextrose or tetramethylurea) resulted in an increase of reagent concentration in the protein-free filtrates or dialysates. This finding is interpreted as a highly selective interaction of water with bovine plasma albumin in the presence of nondenaturants; from this finding, the hydration of bovine plasma albumin in these solvents is calculated to be between 20% to 30% (grams of HOH per g of protein). The increase in concentration of acetamide or ethylene glycol (weak denaturants) in filtrates from solutions containing bovine plasma albumin was found to be dependent on reagent concentration, indicating weak interaction of these denaturants with bovine plasma albumin. In contrast, the concentration of urea or thiourea in filtrates from aqueous solutions decreases following the addition of bovine plasma albumin to strong denaturing solutions of these amides. It is deduced that approximately 1 molecule of urea or thiourea interacts per 3 or 4 amino acid residues of each fully denatured albumin molecule. The extent of interaction of small molecules with albumin, in every case, parallels the ability of the molecules to denature albumin.