Interpretation of cardiac troponin T behaviour in size-exclusion chromatography

Abstract

Background: Knowledge about the presence of intact cardiac troponin T (cTnT) and/or its immunoreactive fragments is of great value for the interpretation of cTnT clearance from the circulation. Until now there has been a lot of controversy about cTnT fragmentation. To provide an answer to this controversy, we investigated fragmentation of cTnT with size-exclusion chromatography (SEC), and confirmed our data using mass spectrometry.

Methods: A highly purified human cTnT standard, characterised using mass spectrometry as a single peak of 34,377 Da and using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) as a single immunoreactive band (37 kDa), was incubated in serum for 0, 24 and 48 h at 37 degrees C and analysed using SEC. A troponin TIC complex standard, used in an earlier study, was also investigated.

Results: We demonstrated that, because of its rod-like shape, the molecular weight of cTnT cannot be estimated from SEC using the molecular weight of globular proteins as a reference. The Stokes radius of intact cTnT was calculated to be 33.7 A. Incubation of both cardiac troponin standards in troponin-free serum resulted in a time-dependent decrease in intact cTnT and a simultaneous increase in smaller immunoreactive fragments (13.4 and 22.4 A).

Conclusions: cTnT has a Stokes radius of 33.7 A. Compared with globular calibrator proteins, intact cTnT elutes earlier than expected based solely on its molecular weight. For non-globular or uncharacterised proteins, Stokes radii should be used for correct interpretation of SEC data. By doing so, we were able to clearly demonstrate cTnT fragments.