Conformational changes at the active site of adenylate kinase detected using a fluorescent probe and monoclonal...

A fluorescent probe, IAEDANS, was introduced into the active site of adenylate kinase (AK) by specifically modifying Cys-25. During modification, enzyme activity was greatly diminished. This probe allowed observation of conformational changes at the active site during denaturation that could not be detected directly in previous studies. The binding ability of modified AK with its monoclonal antibody (McAb3D3) was identical to that of native AK and the fluorescence of modified AK was quenched by interaction with McAb3D3. The relative fluorescence changes during the binding of modified AK with McAb3D3 in different concentrations of guanidine hydrochloride were monitored. The combination of this active site modification with the use of a conformation specific monoclonal antibody has potential for use in the study of the kinetics of folding of AK and in the detection of folding intermediates.