Urease was partially purified from jack bean through four simple steps: aceton extraction-heat treatment-acid precipitation and finally lyophilization.
For extraction one part of meal was mixed with five part of 20% acetone containing 1mM EDTA and 1 mM mercaptoethanol or dithiothrietol and stirred for five minutes.
Concentration of acetone adjusted to 35% and solution brought to 40oC and precipitate was removed by centrifugation. Then urease was precipitated by citric acid and dissolved in phosphate buffer and lyophilized. By this method the purity was increased about 15 fold. The recovery was 60% and yield was 6.5g from one Kg bean. The specific activity was 410 units/mg protein. The molecular weight of the enzyme estimated by gel filtration was 480,000.
In further studies we described an enzymatic colorimetric method for urea determination in body fluids. Urease catalyzed urea hydrolysis and produced ammonia.
Ammonia reacts with glutamine and ATP in the presence of glutamine synthetase and ADP produced. ADP then is assayed in reactions catalysed by pyruvate kinase and pyruvatre oxidase and generate hydrogen peroxide. Hydrogen peroxide is measured at 500 or 550nm.
Aminophenazone is used as the chromogen. The reaction is completed in 15 minutes at 37oC. The standard curve is linear up to 40mmol/L of urea.