Ion-exchange high-performance liquid chromatographic purification of bovine cardiac and rabbit skeletal muscle troponin subunits

Paul J. Cachia, Jennifer Van Eyk, William D. McCubbin, Cyril M. Kay, Robert S. Hodges

Research output: Contribution to journalArticle

Abstract

Bovine cardiac and rabbit skeletal troponin complexes were separated into their respective subunits employing high-performance liquid chromatographic (HPLC) techniques on CM-300 and Q-300 ion-exchangers. Bovine cardiac and rabbit skeletal subunits were separated on the strong anion-exchanger, Q-300, in 8 M urea, 50 mM Tris, 2 mM EGTA, 0.5 mM dithiothreitol, pH 7.5, employing a linear salt gradient and on the weak cation-exchanger, CM-300, in 8 M urea, 50 mM potassium dihydrogen phosphate, 2 mM EGTA, 0.5 mM dithiothreitol, pH 6.5, using a linear salt gradient. To obtain complete purification of all components of troponin both ion-exchangers were required. The initial separation of troponin was carried out on the strong anion-exchanger followed by weak cation-exchange chromatography of the troponin I collected from the strong anion-exchange column. The troponin T subunits obtained from Q-300 chromatography demonstrated heterogeneity (three components: T1, T2 and T3) while the troponin I collected from both sources on the Q-300 column were both resolved into major doublets (I1 and I2) when rechromatographed on the CM-300 column. The three troponin T fractions and two troponin I fractions isolated from ion-exchange HPLC were examined by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis to confirm that the heterogeneity was due to differences in charge and not molecular weight. These results were in agreement with the charge differences observed from retention times on ion-exchange HPLC. When comparing the same troponin subunit from different muscle sources, considerable differences in the content of charged amino acid residues were also observed.

Original languageEnglish (US)
Pages (from-to)315-328
Number of pages14
JournalJournal of Chromatography B: Biomedical Sciences and Applications
Volume343
Issue numberC
DOIs
StatePublished - 1985
Externally publishedYes

Fingerprint

Troponin
Ion Exchange
Troponin I
Purification
Muscle
Ion exchange
Skeletal Muscle
Rabbits
Anions
Urea
Troponin T
Ion exchangers
Dithiothreitol
Egtazic Acid
Liquids
Chromatography
Electrophoresis
Cations
Salts
Ions

ASJC Scopus subject areas

  • Chemistry(all)
  • Clinical Biochemistry
  • Molecular Medicine

Cite this

Ion-exchange high-performance liquid chromatographic purification of bovine cardiac and rabbit skeletal muscle troponin subunits. / Cachia, Paul J.; Van Eyk, Jennifer; McCubbin, William D.; Kay, Cyril M.; Hodges, Robert S.

In: Journal of Chromatography B: Biomedical Sciences and Applications, Vol. 343, No. C, 1985, p. 315-328.

Research output: Contribution to journalArticle

Cachia, Paul J. ; Van Eyk, Jennifer ; McCubbin, William D. ; Kay, Cyril M. ; Hodges, Robert S. / Ion-exchange high-performance liquid chromatographic purification of bovine cardiac and rabbit skeletal muscle troponin subunits. In: Journal of Chromatography B: Biomedical Sciences and Applications. 1985 ; Vol. 343, No. C. pp. 315-328.
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