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 preferentially into the top, PEG-rich, phase of a PEG-salt or PEG-dextran aqueous two-phase system. Extraction by this soluble affinity ligand of proteins is due to a selective interaction of the chelated metal ion with accessible histidine residues on the protein surface. Using Cu-iminodiacetate-PEG (Cu-IDA-PEG) the surface of lactate dehydrogenase (LDH) isoenzymes from different species was probed for the presence of metal chelate binding sites. It was demonstrated that the homotetramers (LDH-1)(H4) from rabbit, bovine and pig displayed weak binding to chelated copper whereas the M4-type isoenzymes (LDH-5) bound strongly to this ligand. The binding of the different heterotetramers increases as the number of M-type subunits increases. In contrast, the human isoenzymes are bound to chelated copper in a reversed sequence. The comparison of the affinity partitioning effect of Cu-IDA-PEG in PEG-salt and PEG-dextran systems revealed that the discriminatory effect of copper is promoted by high salt concentrations. Resolution of isoenzymes by multiple extraction using counter-current distribution provides valuable data on the partitioning of enzymes relative to that of the bulk proteins. The efficacy of metal chelate affinity partitioning for the purification of LDH from tissue samples by batchwise extraction was also demonstrated.
Otto
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A
Institute of Biochemistry, University of Leipzig, Germany.
Birkenmeier
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G
D000818:Animals; D002417:Cattle; D002614:Chelating Agents / Q000737:chemistry*; D002846:Chromatography, Affinity; D003300:Copper / Q000737:chemistry; D003377:Countercurrent Distribution; D003911:Dextrans; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007098:Imino Acids; D007527:Isoenzymes; D007770:L-Lactate Dehydrogenase / Q000032:analysis* / Q000302:isolation & purification; D008670:Metals / Q000737:chemistry*; D009132:Muscles / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology; D011092:Polyethylene Glycols; D011817:Rabbits; D013045:Species Specificity; D013552:Swine
D002614:Chelating Agents; D003911:Dextrans; D007098:Imino Acids; D007527:Isoenzymes; D008670:Metals; D011092:Polyethylene Glycols; D003300:Copper; D007770:L-Lactate Dehydrogenase; C008109:iminodiacetic acid