Crosslinking of troponin complex with 1,3-difluoro-4,6-dinitrobenzene. Identification of the crosslink formed between troponin C and troponin I in the absence of Ca2+.

The single SH-group of rabbit skeletal muscle troponin C (Cys-98) was reacted with the bifunctional reagent, 1,3-difluoro-4,6-dinitrobenzene. This labelled troponin C was used to reconstitute the troponin complex by the addition of equimolar amounts of troponin T and troponin I. The second function of the bifunctional reagent was triggered in the complex by an increase of pH. A crosslink was formed between troponin C and troponin I both in the presence and absence of Ca2+, but the probability of crosslinking was decreased by Ca2+. Covalently linked troponin C-troponin I was isolated from the complex crosslinked without Ca2+, and cleaved by CNBr. The analysis of crosslinked peptides has revealed that in the presence of Mg2+ and absence of Ca2+ the crosslink in the troponin complex is formed between Cys-98 of troponin C and Cys-133 of troponin I.