Details

Autor(en) / Beteiligte

• Wheatley, Jeffrey B.
• Kelley, M.Kathleen
• Montali, Julie A.
• Berry, Christopher O.A.
• Schmidt, Donald E.

Titel

Examination of glutathione S-transferase isoenzyme profiles in human liver using high-performance affinity chromatography

Ist Teil von

• Journal of Chromatography A, 1994-03, Vol.663 (1), p.53-63

Ort / Verlag

Amsterdam: Elsevier B.V

Erscheinungsjahr

1994

Quelle

Elsevier Journal Backfiles on ScienceDirect (DFG Nationallizenzen)

Beschreibungen/Notizen

• A method for the examination of the glutathione S-transferase isoenzyme profiles in human liver using a new HPLC affinity support is described. Liver cytosol was injected directly onto an HPLC column (5 × 0.46 cm) containing a support with a covalently bound affinity ligand (S-octylglutathione) specific for the isoenzymes. Contaminating cytosolic proteins were removed in a washing step. The isoenzymes were eluted with a linear gradient of a different affinity ligand in the mobile phase. Coinciding with the affinity ligand gradient, a salt gradient (0–200 m M sodium chloride) was applied. In this manner the isoenzymes were fractionated into the enzymatically active homodimers and heterodimers. The classes of the affinity fractionated isoenzymes were determined by SDS-PAGE and ELISA while the subunit content was determined by reversed-phase chromatography. For one liver three Alpha class isoenzyme subunits, forming three heterodimers and two homodimers, were detected. Five livers were examined, and the homodimer A1-1 was found to be the predominant glutathione S-transferase isoenzyme. Minor amounts of Pi and Mu class isoenzymes were also detected. This non-denaturing high-performance affinity chromatography method reduced analysis time by a factor of ten when compared to other affinity analysis methods for the glutathione S-transferases.