I performed BApNA assay for soybean samples. I also took the readings using spectrophotometer. But how should I calculate the TUI for the same samples?
You can not take trypsin in blank. without PI OD will be more and with PI OD will be less. First you calculate trypsin activity without PI, consider it as 100% and then calculate PI activity by considering reduction in OD.
To calculate the TUI (Trypsin Units per mg of protein inhibition) for your soybean samples, you first need to determine the trypsin activity in the presence and absence of the trypsin inhibitor. Here are the steps:
Calculate the trypsin activity in the control and samples:
For the control, subtract the absorbance reading of the blank from the absorbance reading of the control. This gives you the net absorbance (A).
Convert the net absorbance to trypsin activity using a standard curve of known trypsin concentrations. The equation for the standard curve can be derived from the Michaelis-Menten equation.
For the samples, subtract the absorbance reading of the blank from the absorbance reading of the sample. This gives you the net absorbance (A).
Convert the net absorbance to trypsin activity using the same standard curve.
Calculate the trypsin inhibition in the samples:
Subtract the trypsin activity in the presence of the trypsin inhibitor from the trypsin activity in the absence of the inhibitor. This gives you the trypsin inhibition (I).
Calculate the TUI using the following formula:
TUI = (I / mg of protein) x (1 / V)
where:
mg of protein: the amount of protein in the sample (in mg)
V: the volume of the sample extract used (in mL)
Note: You need to measure the protein content of your sample separately, using a protein assay such as the Bradford assay or the Lowry assay.
In summary, the TUI is calculated as the amount of trypsin inhibited per mg of protein in the sample extract.
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