ИСТИНА

Background. Cardiac troponin I (cTnI) is a golden marker of myocardial cell death. It was shown that a wide diversity of cTnI forms, including proteolytic fragments could be found in patients’ blood. Partial cTnI proteolysis happens from N- and C-terminal parts of the molecule, whereas central part of cTnI remains relatively stable. Though N- and C-terminal degradation of cTnI was described more than 15 years ago, still it is not clear, how it happens - is it a sequential truncation, or relatively big fragments from both ends are cleaved on the very first steps of cTnI degradation. Methods. Cardiac proteins were extracted from human cardiac tissue and incubated at 37ºC for 3 hours. In the preliminary experiments it was shown that in such conditions cTnI is partially cleaved by co-extracted endogenous proteases. After incubation extracted proteins were separated by gel-filtration (GF) chromatography. cTnI immunoreactivity in GF fractions was measured by sandwich immunoassays utilizing pairs of antibodies specific either to N- or to C-terminal parts of the molecule. On the next step cTnI fragments from GF fractions were purified by means of affinity and reverse-phase chromatography. Purified fragments were analyzed by mass spectrometry. Results. Both types of immunoassays (specific to N- and C-terminal parts of cTnI) revealed two peaks of immunoreactivity in GF fraction. In both cases the first peak was detected in fractions corresponding to the proteins with molecular masses about 60-80 kDa (most likely cTnI in ternary complex), whereas second peak of immunoreactivity was found in fractions corresponding to the proteins with significantly lower molecular masses. By MS studies we were able to identify only few peptides (27-36 amino acid residues long) truncated from the N-terminal part of the molecule and multiple peptides (20-40 amino acid residues long) from the C-terminal part. Peptide sequence analysis suggested that the N-terminal part of cTnI has a very limited number of cleavage sites, whereas C-terminus contains much more sites of protease degradation. Conclusion. Using different biochemical methods and mass spectrometry analysis we were able to purify and identify peptides that are formed in vitro after cTnI cleavage by endogenous proteases. Our studies suggest that relatively big and relatively stable peptide(s) is (are) truncated from the N-terminal part of cTnI. C-terminus of cTnI molecule contains multiple possible sites of proteolysis and protease cleavage results in formation of wide diversity of different size peptides.