Collect. Czech. Chem. Commun. 1989, 54, 2814-2822

Affinity chromatography of arginase with continuous detection of enzyme using urease membrane electrode

Lumír Macholán and Bohdana Hlavatá

Department of Biochemistry, J. E. Purkyně University, 611 37 Brno


A rapid method is described of potentiometric measurement of liver arginase during its purification on a column of affinity adsorbent prepared by the derivatization of Spheron E-1000. The detection of the enzyme in the flow-through system is effected by an ammonia electrode coated by the reaction layer of glutaraldehyde-crosslinked urease on a polyamide mesh. The method is time- and urease-saving and permits arginase to be obtained for analytical purposes purified 212 times from a liver extract in a 68% yield. By combining the membrane made of crosslinked arginase and urease on a polyamide mesh with a pNH3 electrode we developed a two-enzyme sequence electrode sensitive to L-arginine (10-3 -10-4 mol l-1) showing a response time of 1-3 min and a stability of about 3 weeks.