IDENTIFICATION OF REACTIVE CARBOXYL GROUPS IN TROPONIN-C

Ca2+ regulates muscle contraction by reversible binding to troponin C (TnC), the Ca2+-binding subunit of troponin complex. In order to ident ify acidic amino acids exposed on its surface, carboxyl groups in TnC were activated with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, th en labeled with dinitrophenylethylenediamine. Labeled protein was then digested with trypsin and thermolysin, and the resulting peptides wer e purified by HPLC. Modified peptides were detected by their specific absorbance at 360 nm, and labeled amino acids in these peptides were i dentified by sequence analysis. Although the total incorporation of la bel 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 wer e located in surface regions of the known three-dimensional structure of TnC which may interact with other components of the troponin comple x.