Effect of denaturing agents of the urea-guanidinium class on the solubility of acetyltetraglycine ethyl ester and related compounds.

It has been demonstrated by several investigators, most convincingly by studies of the effect of urea on the solubility of various compounds, that aqueous urea solutions are better solvents than pure water for many solutes containing hydrophobic groups (l-5). It has further been suggested that this property of aqueous urea solutions is responsible for their denaturing activity toward proteins and deoxyribonucleic acid (l-5). There is evidence supporting this hypothesis for the denaturation of DNA and some evidence suggesting that this property may contribute to the denaturing activity of compounds of the ureaguanidinium class toward certain proteins (5, 6). On the other hand, the absence of a positive correlation between the hydrophobic character and the denaturing effectiveness of denaturing agents toward bovine serum albumin and ovalbumin indicates that other properties of these reagents must contribute to their denaturing activity (6-9). The effects of substances of the ureaguanidinium class on the solubility of model compounds, which are reported in this communication, demonstrate that this class of denaturing agents can promote protein denaturation by causing a decrease in the activity coefficient of the peptide group. This effect is not due to an increase in the solvent power of the solutions toward hydrophobic solutes; i.e. it is not a “hydrophobic” effect in the sense that this term is ordinarily used. N-Acetyltetraglycine ethyl ester, a model compound composed largely of amide groups, was prepared from tetraglycine ethyl ester hydrochloride (10) and acetic anhydride in 40% aqueous pyridine at 0”. The product crystallized from the reaction mixture; after one recrystallization from water it had a melting point of 263-264’ (with decomposition).