I am working on enzyme and its assay method is HPLC. So I have two things to monitor 1) Decrease in substrate concentration (area under curve of substrate) after specific time 2) Increase in product concentration (area under curve of product) after specific time. From the area under curve I can monitor the activity of enzyme but could not able to calculate enzyme activity in U/ml.
Please do needful.
Hi Dattatray,
You would have to run a standard curve of known substrate and product concentrations to assess the area under the curve for each one of the concentrations. This will enable you to convert the variable "area" into known concentrations and will facilitate your calculation of substrate consumed or product formed in the test reaction that you are running. Kindly see the below mentioned paper where I ran a ion-pair reverse phase HPLC to assess my guanosine formation and GMP hydrolysis and quantified subsrate consumed/product formed by quantifying the area under the curve:
Srinivasan B et al ( 2014) FEBS J. 281(6), 1613-1628.
I hope my answer helps.
Hello Bharath Sir,
Thanking you for your attention to my question. I have already run the standard curve of both substrate and product and converted area under curve into concetration of comsumed substrate and form product. My query is how to calculate enzyme activity from the respective concentration of substrate/ product. How we will get activity in U/ml? Is there any formula to calculate the same? I gone through your research article but still couldnt find any formula.
I hope you understood my query.
Hi Dattatray,
Thats should be pretty straighforward. If you know the concentration of your substrate or product, the volume of your reaction mix injected in the HPLC and the time at which you quenched your reaction, you can back calculate "U". "U" of enzyme activity/ml has a specific definiton like "1 unit (U) is the amount of enzyme that catalyses the reaction of 1 micro or nano mol of substrate per minute". Since you know the time, the concentration and the volume, you can use direct proportionality to calculate "U/ml". There is no specialized formula as such. I hope my answer is helpful.
Remember, 1 enzyme molecule can convert 1000's of substrate molecules before it is exhausted. The response from an HPLC is mass related. The 2 can be related but it depends on other parameters like pH, time, substrate concentration, sample matrix....
Remember that the official unit for enzyme activity is NOT the U (unit) (millimol per min) but the KATAL (mol/s)
Well by HPLC you are checking the amount of substrate converted by the enzyme and therefore, there is no separate method of enzyme calculation for using such amethod. The basic concept that the activity of the the enzyme is : amount of protein required to convert unit substrate in unit time; remains the same.
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