Trypsin and chemotrypsin colorimetric assay

Dr Engelbert Buxbaum via methods%40net.bio.net (by engelbert_buxbaum from hotmail.com)
Sat May 19 11:37:09 EST 2012


In article <mailman.235.1335815731.3456.methods from net.bio.net>, 
Gerchman from research.haifa.ac.il says...
> 
> I am looking for recommendations for Trypsin and
> Chemotrypsin colorimetric assay, preferably not in the UV range. 

The classical assay is with azocasein  (e.g. Charney and Tomarelli, JBC 
171 (1947) 501), this colorimetric assay is read at 440 nm. 

More modern assays work with Förster's resonance energy transfer: the 
protein is labeled with two fluorescent dyes in resonance (emission 
spectrum of the first overlaps the absorption spectrum of the second). 
As the protein is digested, distance between the dyes increases and 
resonance (which decreases with the 6th power of the distance) is 
abolished. The advantage is that you do not need to separate hydrolyzed 
product from non-hydrolyzed substrate and can measure while the reaction 
is occurring. Therefore, this type of assay is suitable for large-scale 
screening.


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