Inter- and intramolecular interactions of alpha-lactalbumin. V. The effect of amidination on association and aggregation.

Abstract

Abstract 1. 1. α-Lactalbumin exists as the monomeric unit at pH values alkaline to the isoelectric point (4.25-4.50). However, below pH 4.00 the protein undergoes a confirmational change which results in strong association and aggregation. To determine if hydrophobic interactions play a role in the association and aggregation, new nonpolar groups have been introduced into the protein by the amidination reaction using ethyl acetimidate hydrochloride and ethyl butyrimidate hydrochloride. 2. 2. A comparison of the properties of the modified proteins with α-lactalbumin by sedimentation velocity, ultraviolet fluorescence, optical rotatory dispersion and electrophoresis showed that they retain most of the characteristics of α-lactalbumin indicating that no major structural changes have resulted from the modification. It was found, however, that the modified proteins were much more susceptible to association and aggregation than α-lactalbumin. This was evident not only at pH 2.00 but on the alkaline side of the isoelectric point at pH 6.00 and 8.50. These observations lend support to the hypothesis that hydrophobic interactions play a role in the association and aggregation of α-lactalbumin and that the difference in behavior of the “alkaline” and “acid” forms of the protein are due to a higher density of non-polar groups at the molecular surface of the “acid” form.