Identification of reactive carboxyl groups in troponin C

Ca2+ regulates muscle contraction by reversible binding to troponin C (TnC), the Ca(2+)-binding subunit of troponin complex. In order to identify acidic amino acids exposed on its surface, carboxyl groups in TnC were activated with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, then labeled with dinitrophenylethylenediamine. Labeled protein was then digested with trypsin and thermolysin, and the resulting peptides were purified by HPLC. Modified peptides were detected by their specific absorbance at 360 nm, and labeled amino acids in these peptides were identified by sequence analysis. Although the total incorporation of label into TnC was only 2.6 mol/mol, we found that 14 of the 46 carboxyl groups of TnC were partially labeled. The labeled carboxyl groups were located in surface regions of the known three-dimensional structure of TnC which may interact with other components of the troponin complex.