RECOGNITION AND SEPARATION OF ISOENZYMES BY METAL-CHELATES - IMMOBILIZED METAL-ION AFFINITY PARTITIONING OF LACTATE-DEHYDROGENASE ISOENZYMES

Poly(ethylene glycol) (PEG)-bound chelated metal ions partition prefer entially into the top, PEG-rich, phase of a PEG-salt or PEG-dextran aq ueous two-phase system. Extraction by this soluble affinity ligand of proteins is due to a selective interaction of the chelated metal ion w ith accessible histidine residues on the protein surface. Using Cu-imi nodiacetate-PEG (Cu-IDA-PEG) the surface of lactate dehydrogenase (LDH ) isoenzymes from different species was probed for the presence of met al chelate binding sites. It was demonstrated that the homotetramers ( LDH-1)(H-4) from rabbit, bovine and pig displayed weak binding to chel ated copper whereas the M4-type isoenzymes (LDH-5) bound strongly to t his ligand. The binding of the different heterotetramers increases as the number of M-type subunits increases. In contrast, the human isoenz ymes are bound to chelated copper in a reversed sequence. The comparis on of the affinity partitioning effect of Cu-IDA-PEG in PEG-salt and P EG-dextran systems revealed that the discriminatory effect of copper i s promoted by high salt concentrations. Resolution of isoenzymes by mu ltiple extraction using counter-current distribution provides valuable data on the partitioning of enzymes relative to that of the bulk prot eins. The efficacy of metal chelate affinity partitioning for the puri fication of LDH from tissue samples by batchwise extraction was also d emonstrated.