Hello everyone!
I have done an experiment in enzyme assay of alpha glucosidase by using pNPG ( p- nitrophenol-a-D-glucopyranoside ) as a substrate. This is my mixture reaction: 0.9ml phosphate buffer, 0.1 ml enzyme solution. After 10 min of reaction, I add 1ml Na2CO3 1M to stop the reaction and measure the absorbance of p-nitrophenol released by employing spectrophotometer (Abs 400nm). I got the OD values but how can I use these figures to calculate enzyme activit (Unit - U)?
Thank you for reading my question and I really appreciate if you can help me!!!
Hi there,
First of all you have to check that OD does vary linearly with the time. In order to do so you should repeat the experiment with arrest of the reaction at different times between 0 and 10 min. By plotting OD versus time you should be able to trace a straight line. The slope is the velocity of the reaction expressed variation of OD per time unit. To convert this into unit (1 unit being one µmol/min) you need to use Beer's law to get velocity expressed in terms of concentration/min then you have to double this concentration as arrest is made by 2 time dilution of reaction mixture. Then multiply by volume 1mL to get velocity expressed as mol/min. Then convert it into µmol/min. Knowing that this velocity was obtained using 0.1mL of enzyme solution then you may express enzyme activity as U/mL by dividing velocity by 0.1. Activity is expressed as U/mL of enzyme solution.
Thank you Dominique Liger , I'm trying to calculate it with your help. Really appreciate your great help!!
Simply you can use p-Nitrophenol as standard for this test. then take different concentrations of Nitrophenol and follow the same assay protocol and you will come to compare your OD (sample) with the OD of standard, i.e., Nitrophenol....then you can calculate the Enzyme activity by plotting a standard graph and putting your OD in Y- equation obtained from the standard...
Hope it helps....
Why are you doing an end-point determination? If you measure the abs at 400 nm continiously you will directly see if you measure the initil rate (linear part) and your slope will be more accurate.
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