Ammonium Carbamate as the Intermediate Product in the Action of Urease on Urea

Abstract

A method for quantitative estimation of ammonia nitrogen and carbamic acid nitrogen, in a solution containing ammonium salts, urea, and carbamates, has been developed. The method depends on the fact that ammonium carbamate is stable in Nessler solution so that one-half of the nitrogen in the ammonium carbamate does not affect the color of the Nessler solution. However, if the solution is acidified before Nesslerizing, the carbamic acid is almost instantly decomposed and Nesslerization gives the total of ammonia and carbamate nitrogen present. In other particulars analyses are made according to the well-known colorimetric method of determining ammonia. Each solution to be analyzed was Nesslerized under these 2 different conditions. The difference between the two values for nitrogen was equal to the carbamic acid nitrogen. Twice this figure was equal to the ammonium carbamate nitrogen. The method was shown to be quantitative within the limits of accuracy of reading a colorimeter. A number of experiments have been carried out in which the enzyme was allowed to act on 100 cc. ice-cold 1% urea. The action was stopped after 5 to 7 minutes by adding a trace of potassio-mercuric iodide. During this time about 1 mg. per cc. of total combined nitrogen had been liberated from the urea. This concentration was found to be most convenient for analysis. After poisoning the enzyme, analyses were made immediately for ammonium carbamate and ammonium carbonate and then at intervals until the carbamate had fallen almost to zero. Figure 1 shows the curve for percentage ammonium carbamate nitrogen of the total nitrogen when plotted against time after the enzyme had been stopped. The percentage of carbamate diminished very rapidly but at a measurable rate. Our method of analysis was not accurate enough to determine the point of equilibrium between the carbonate and the carbamate.