Calculation of Chymotrypsin units inhibited per mg sample

[gbala87]

Hi,

I have trouble calculating CI/mg sample for my inhibitor. To brief the procedure, the inhibitor solution was diluted appropriately so that 1 ml will give 40-60% inhibition (In this case the stock solution was 0.4 mg/ml). In a 1 ml assay volume, 0.2 ml of inhibitor sample (by taking different volumes of stock solution: 0.05 ml (0.02 mg), 0.1 ml (0.04 mg), 0.15 ml (0.06 mg) & 0.2 ml (0.08 mg) is added with 0.2 ml of chymotrypsin solution (0.04 mg/ml) and then 0.5 ml of substrate (GPNA). After 10 min incubation at 37 deg C, 0.1 ml of 30% acetic acid was added to terminate the reaction. The samples were centrifuged and read at 410 nm. % chymotrypsin inhibition was calculated after correcting with sample and reference blanks and below is the respective values for above mentioned concentrations 38.3%, 58.3%, 66.7% and 75%.
As per the procedure, the graph is plotted for concentration vs % inhibition values and then concentration for 100% inhibition is extrapolated from the graph. After calculating from the graph, the concentration for 100% inhibition is come to 0.11 mg. I hope I am right up to this point. Next step is to calculate 1 mg of Chymotrypsin inhibited per mg of inhibitor and then I should convert this to mg of Chymotrypsin inhibited per gram of product to get CI Activity.

Hope I made my case clear.
Thanks in advance.

[ChenBeier]

You described the procedere of sample preperation and measurement. I think it's ok, but what is your question?
So far for 0.2 ml Chemotrypsin you need 0.11 mg Inhibitor. Question is the inhibitor caralytic or do you need more according concentration of Chemotrypsin.

[gbala87]

Hi,

My question is how can I use the calculated values to express the final activity of my inhibitor in mg of chymotrypsin inhibited per mg of inhibitor?

[ChenBeier]

The calbration curve is made with a const. concentration of 0,2 ml = 0,008 mg Chemotrypsin.
Changed were the amount of inhibitor.
Different amount of inhibitor gives different activities.
But we don't know if amount of Chemotrypsin is changed to 1 mg the activities goes with the increase.
I think you need a curve with a constant amount of inhibitor and different amount of Chemotrypsin.

[JustinPlaying]

To calculate the CI (chymotrypsin inhibition) activity per mg of inhibitor and then convert it to mg of chymotrypsin inhibited per gram of product, follow these steps:
Step 1: Summary of Your Data
1. Inhibitor Concentrations:
2. Chymotrypsin Concentration:
3. Extrapolated Concentration for 100% Inhibition:

Step 2: Calculate Chymotrypsin Inhibited at 100% Inhibition
At 100% inhibition, all the chymotrypsin present in the assay is inhibited. Therefore, the amount of chymotrypsin inhibited is equal to the total amount of chymotrypsin in the assay:
Chymotrypsin inhibited=0.008mg

Step 3: Calculate CI in mg of Chymotrypsin Inhibited per mg of Inhibitor
Inhibitor Amount for 100% Inhibition:

The amount of inhibitor corresponding to 100% inhibition is 0.11 mg.

Step 4: Convert CI to mg of Chymotrypsin Inhibited per Gram of Product
This method should give you the correct calculations based on the data provided

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