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I have to determine the amount of urea in urine for a biochemistry investigation.
Equation: Urea + water + urease enzyme -> ammonium carbonate
In a method for making a calibration curve which I got from a book, it says to add the 2 cm3 x% solution of urea to 50 cm3 of 4% solution of urease (enzyme) and leave in a waterbath maintained at 35 oC for an hour.
I carried out a preliminary investigation, but my school only allowed me to use 10 cm3 5% urease (enzyme) solution to 2 cm3 x% urea solution. As the pH of ammonium carbonate is approximate, I used universal indicator to see when the reaction was completed. I found it was completed within 10 minutes, which is a pretty small value compared with one hour.
Do you have an ideas why the method states 1 hour? Am I missing something fairly obvious?
020608
PJ writes ...
I'm surprised both at the quantities of enzyme solution required (50 cm3 of a 4% enzyme solution is a lot of enzyme) and at the length of incubation time. Sounds very suspicious to me.
Urease is a rapid and robust enzyme, and I would say that 10 minutes sounds ample for a significant amount of reaction to take place.
Here is a method, measuring urease activity, that appeared in the Nuffield A-level Chemistry Students Book (3rd edition, 1994), p408/409, experiment 14.4c, which could be adapted to measure urea in urine.
In this method, 5 drops of full range indicator are added to 5 cm-3 of 0.25 mol dm-3 urea, followed by sufficient drops of 0.01 mol dm-3 HCl to just turn the indicator red. 1 cm3 of 1% urease active meal (to which indicator and acid have been similarly added) is added, and the time for the colour of the indicator to change (due to the production of ammonia) is found. A very neat method. And it works!
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updated: 08 June 2008
