Amide proton titration shifts in bull seminal inhibitor IIA by two-dimensional correlated 1H nuclear magnetic resonance (COSY). Manifestation of conformational equilibria involving carboxylate groups.

Amide proton titration shifts in H2O solution of bull seminal inhibitor IIA were measured over the pH range from 3 to 6 using two-dimensional correlated spectroscopy. These data enabled characterization of the pKa values for the majority of the carboxylate groups in the protein. Two glutamate side-chains were found to form hydrogen bonds with their own backbone amide proton. Different temperature variations of the populations of these local, cyclic structure elements are indicated for the individual sites.