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Biotechnology and Applied Biochemistry (2001) 33, (107–115) (Printed in Great Britain)

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Biochemical and immunological properties of human cardiac troponin I fragments

Nihmat Morjana¹, Douglas Clark and Rony Tal²

Dade Behring Inc., P.O. Box 6101, Glasgow, DE 19714, U.S.A.

Abbreviations used: CK-MB, creatine kinase that contains the M and B subunits; DTT, dithiothreitol; Endo-Asp, endoproteinase Asp-N; MI, myocardial infarction; Ni-NTA, Ni²⁺-nitrilotriacetate; TnC, troponin C; TnI, troponin I; TnT, troponin T; cTnI, cardiac TnI; rTnI, recombinant TnI.

¹ To whom correspondence should be addressed (e-mail: morjanna@dadebehring.com).

² Present address: Sunol Molecular 2810 North Commerce Parkway, Miramar, FL 33172, U.S.A.

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 (TnI₁₅₃). Cleavage of the rTnI with the endoproteinase Asp-N generates a smaller TnI fragment (TnI₈₈, residues 6–96). TnI₁₅₃ has higher immunological activity than that of rTnI and lower activity than that of TnI₈₈, as judged by the Stratus II^® TnI Immunoassay. TnI₁₅₃ 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 TnI₁₅₃, but has little effect on the activity of TnI₈₈. The TnI₁₅₃–TnC complex exhibits higher immunological activity than rTnI–TnC and TnI₈₈–TnC, and much higher activity than free rTnI, TnI₁₅₃ and TnI₈₈. The presence of TnT has no effect on the immunological activity of the TnI₁₅₃–TnC complex, suggesting that the addition of TnT does not interfere with TnI₁₅₃ recognition by TnI monoclonal antibodies. Free TnI₁₅₃ and TnI₈₈ degrade rapidly in human serum. TnC protects TnI₁₅₃ from proteolytic degradation, but offers less protection for TnI₈₈. The TnI₈₈–TnC complex lost 80% of its immunological activity after incubation for 2 days in human serum at 37 °C. However, there was no loss in the immunological activity of the TnI₁₅₃–TnC complex under the same conditions. A cTnI fragment (TnI₈₀, residues 1–80), expressed in E. coli as a fusion protein, exhibits immunological activity and stability similar to that of TnI₈₈.

Received 24 November 2000; accepted 2 January 2001

Portland Press Ltd © 2001