A new purification procedure for fumarase based of affinity chromatography. Isolation and characterization of pig-liver fumarase.
A new procedure has been developed for the isolation of fumarase (EC 4.2.1.2). It is described for the purification of pigheart and liver enzyme. Pyromellitic acid has been covalently coupled to Sepharose-4B with diaminopropanol as spacer arr. When a dialysed 0.55 saturated ammonium sulphate precipitate is applied to the column, in Tris-acetate buffer, pH 7.3, fumarase remains quantitatively bound. It is eluted by competition, together with a few other proteins, by the natural product L-malate. Malate is removed from the eluate by dialysis. After this highly efficient purification step the enzyme is very easily crystallized. The final yield by 67% for both pig heart and liver preparations. The specific activity of fumarase purified from both tissues is found to be the same. Polyacrylamide gel electrophoresis in dodecylsulphate shows one single band corresponding with a subunit molecular weightof 48500. A single band is also obtained by electrophoresis in acid urea. This new procedure based on biospecific affinity chromatography allows a fast and easy preparation of gram quantities of fumarase.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001565:Benzoates / Q000494:pharmacology; D002846:Chromatography, Affinity; D005649:Fumarate Hydratase / Q000302:isolation & purification* / Q000378:metabolism; D007461:Iodoacetates / Q000494:pharmacology; D007700:Kinetics; D008024:Ligands; D008099:Liver / Q000201:enzymology*; D009206:Myocardium / Q000201:enzymology; D009928:Organ Specificity; D010071:Oxaloacetates / Q000494:pharmacology; D013552:Swine
D000596:Amino Acids; D001565:Benzoates; D007461:Iodoacetates; D008024:Ligands; D010071:Oxaloacetates; C012018:pyromellitic acid; D005649:Fumarate Hydratase