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.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002848:Chromatography, DEAE-Cellulose; D002852:Chromatography, Ion Exchange; D003488:Cyanogen Bromide / Q000494:pharmacology; D004139:Dinitrofluorobenzene / Q000031:analogs & derivatives / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000378:metabolism; D009132:Muscles / Q000032:analysis; D009578:Nitrobenzenes / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis; D011817:Rabbits; D013053:Spectrophotometry; D014336:Troponin / Q000378:metabolism*; D019209:Troponin C; D019210:Troponin I
D000596:Amino Acids; D009578:Nitrobenzenes; D010446:Peptide Fragments; D014336:Troponin; D019209:Troponin C; D019210:Troponin I; D004139:Dinitrofluorobenzene; C002041:1,5-difluoro-2,4-dinitrobenzene; D008274:Magnesium; D003488:Cyanogen Bromide; D002118:Calcium