Biochemical and immunological properties of human cardiac troponin I fragments

Abstract

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 degrees 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.

Heterogeneity in Human Cardiac Troponin I Standards

“…Accordingly, peptides containing the two cysteine residues were observed at masses that indicate alkylation by 2-mercaptoethanol, as noted previously for the intact protein. Two peptides were observed with molecular masses that correspond to fragments [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] and with an additional 563 u. If their assignment is correct, these peptides verify the presence of the expression tag on the N-terminus of this recombinant protein.…”

“…One peptide observed with a molecular mass of 1975.8 Ϯ 1 could be assigned as fragment [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19], whose calculated molecular mass of 1976.1 includes acetylation on the Nterminus. No peptides were observed at a mass corresponding to [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] without acetylation. Peptides were observed at molecular masses that agreed well with the calculated molecular masses of peptides and [1-39], with both peptides being N-acetylated and containing two phosphate groups.…”

“…After myocardial infarction, cTnI is released into the bloodstream from damaged heart muscle tissue. Elevated levels of cTnI can be found in the bloodstream for as long as 7 days, with concentrations peaking 18 -24 h after the onset of the infarction (1). Unlike many other protein markers for myocardial infarction, such as creatine kinase MB (CK-MB), cTnI is heart--specific.…”

Mechanism for stabilization of the molten globule state of papain by sodium n-alkyl sulfates: Spectroscopic and calorimetric approaches

“…The presence of low concentrations of GuHCl could stabilize the molten globule state of apomyoglobin and cytochrome c [13]. GuHCl-induced stabilization of protein is also seen with disulfide isomerase [14], suggesting that chaotropic agents at lower concentrations can stabilize the partially folded intermediates though they behave as potent denaturants at higher concentrations. Further, it has been shown that n-alkyl sulfates also stabilize intermediate states in the folding/unfolding pathway of cytochrome c [15][16][17][18].…”

Spiegelmers as potential receptors for cTnI diagnostics

“…It is well documented that cTnI is prone to degradation and binding to plastic and glass surfaces. 15,16 Therefore, the dilution linearity was analysed by determining the cTnI concentration of spiked specimens with a clinically applied, two-step chemiluminescence immunoassay, which is distributed by ARCHITECT under the brand name STAT Troponin-I. In the rst step, cTnI binds to antibody-coated paramagnetic microparticles.…”