Biochemical and immunological properties of human cardiac troponin I fragments

Cardiac troponin I (cTnI) is the inhibitory subunit of the troponin complex and is a biochemical marker for myocardial infarction (MI). It is found in human serum within 4-6 h following MI. One of us has shown [Morjana (1998) Biotechnol. Appl. Biochem. 28, 105-111] that MI patient serum TnI is cleaved at the N- and C-terminals and that the TnI fragments exist as a complex with tropinin C (TnC) and troponin T (TnT). In the present study, we have generated C-terminal truncated TnI fragments and studied their immunological and biochemical properties. Human recombinant TnI (rTnI) expressed in Escherichia coli is cleaved into a major fragment with a molecular mass of 17500 Da using CNBr. The major CNBr fragment contains the first 153 amino acids of human cTnI (TnI(153)). Cleavage of the rTnI with the endoproteinase Asp-N generates a smaller TnI fragment (TnI(88), residues 6-96). TnI(153) has higher immunological activity than that of rTnI and lower activity than that of TnI(88), as judged by the Stratus II(R) TnI Immunoassay. TnI(153) exhibits biochemical and immunological properties similar to those of intact TnI. It binds TnC at a molar ratio of 1:1 and forms a ternary complex with TnC and TnT. TnC enhances the immunological activity of TnI(153), but has little effect on the activity of TnI(88). The TnI(153)-TnC complex exhibits higher immunological activity than rTnI-TnC and TnI(88)-TnC, and much higher activity than free rTnI TnI(153) and TnI(88). The presence of TnT has no effect on the immunological activity of the TnI(153)-TnC complex, suggesting that the addition of TnT does not interfere with TnI(153) recognition by TnI monoclonal antibodies. Free TnI(153) and TnI(88), degrade rapidly in human serum. TnC protects TnI(153) from proteolytic degradation, but offers less protection for TnI(88). The TnI(88)-TnC complex lost 80% of its immunological activity after incubation for 2 days in human serum at 37 degreesC. However, there was no loss in the immunological activity of the TnI(153)-TnC complex under the same conditions, A cTnI fragment (TnI(80), residues I-80), expressed in E. coli as a fusion protein, exhibits immunological activity and stability similar to that of TnI(88).