Coulometric Flow Analyzer for Use with Immobilized Enzyme Reactors

A flow coulometric immobilized enzyme analyzer based on the use of a fast totally electrochemical pH-stat has been developed. The insolubilized urease had a Km of 43 mM and an activity of 1000-1200 units per gram of glass at its pH optimum of 6.8 in 0.2 M sodium perchlorate. In the absence of phosphate buffer, urea adsorbed on the immobilized enzyme, causing broad peaks. Peak width was decreased by adding 1,3-diaminopropane to the electrolyte. It serves as a competitive inhibitor (K1 = 79 µM) and blocks adsorption of urea. Urea could be analyzed with good precision (3%) in simulated sera and in quality control reference sera. Recoveries of urea added to quality control sera were 100%. The major problem involved in determining urea in human serum was a high and irreproducible blank which limited the precision to about 5%.