Hello
I want determine degree of hydrolysis by measure the percentage of protein soluble in 10 % trichloroacetic acid /total protein in sample, how I can measure the total protein with biuret method? can I calculate the protein of supernatant of reaction mixture and use it as total protein of sample?
The biuret assay is performed at basic pH, so you will have to neutralize the TCA before measuring the protein content of the supernatant. You can probably also resuspend the pellet in the assay reagent to measure its protein content. The sum of the pellet and supernatant protein contents should equal the total amount of protein that was present in the protein at the start, including the proteolytic enzyme.
I am not sure what you mean by measuring total protein, this you should know as independent variable.
To measure protease activity there is a classic method using azocasein as substrate. After TCA precipitation of non-hydrolysed protein the TCA-soluble azo-peptides can be determined colorimetrically in the supernatant:
@ARTICLE{Haz-65,
author = {Hazen, G.G. and Hause, J.A. and Hubicki, J.A.},
title = {An automated system for the quantitative determination of proteolytic enzymes using azocasein},
journal = {Ann. N.Y. Acad. Sci.},
volume = {130},
number = {2},
doi = {10.1111/j.1749-6632.1965.tb12620.x},
pages = {761-768},
year = {1965},
}
@BOOK{Haz-74,
AUTHOR = {G.G. Hazen},
EDITOR = {H.U. Bergmeyer},
BOOKTITLE = {Methods of enzymatic analysis},
YEAR = {1974},
PUBLISHER = {VCh},
address = {Weinheim},
volume = {2},
pages = {1000-1005},
}
Modern fluorimetric methods (e.g., https://www.thermofisher.com/de/de/home/life-science/cell-analysis/fluorescence-microplate-assays/microplate-assays-enzyme-activity.html) are probably more sensitive, but this one still climbs the hill.
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