Hello Nisha Reghu

Proteases are enzymes, naturally present in all living cells and tissues, involved in a multitude of physiological reactions that includes digestions of proteins in the food to highly regulated cascades. Proteases can either break specific peptide bonds, depending on the amino acid sequence of a protein or completely breakdown peptides to amino acids. In certain physiological conditions, protease activity can abolish a protein's function or an activation of a function.

For studying proteins, tissues and cells are lysed, which release proteases in the sample and their activities can be measured using a protease assay. Monitoring various protease activities has become a routine task for many laboratories. Some proteases have been identified as new drug development target.

Principle of protease assay:

Proteases catalyze hydrolysis of the peptide bond and therefore break proteins into small fragments. In this assay, the substrate casein is hydrolyzed to release tyrosine and tryptophan, which then react with Folin’s reagent to produce chromophores giving a blue color. The color can be measured by a spectrophotometer at 680nm to quantify the amount of casein that is hydrolyzed. A standard curve is necessary to correlate the intensity of the color with the amount of tyrosine and further with the protease activity.

Best.

There are many, many different proteases, and numerous methods to measure their activity. Can you be more specific?

Protease is an protein degrading extracellular enzyme . The source for the isolation of protease is any living organism, so depending upon the species specific its variation in assays per-formation. But no dout the common substrate is used as a casein to show the enzyme activity.

Please mention the name of source from which you see the protease activity

The answers above detail what a protease is and how to measure it. But why do we care to measure this? Some people want to identify proteases in their protein solutions either to get rid of them or to see what type they are. Some scientists work with specific proteases and therefore want to check for their presence AND their activity. There are many assays to do this. If you know what protease you are looking for, the assay can be made specifically for that enzyme.

Proteases are enzymes that degrade proteins, that is, chop them up in smaller pieces. The speed with which the pieces apear is the protease activity, measured in mol/s = katal. In the older literature you may also find the non-SI unit U = micromol/min.

One classic assay is to pellet the protein (for example azo-casein) with TCA and measure the concentration of soluble fragments. Another uses FRET: the protein is labelled with two fluorescent dyes which are in resonance (that is, the emission wavelength of the first is close to the excitation wavelength of the second). As long as both dyes are close together, the excited first dye will transfer its energy to the second, and its fluorescence is not detectable. If the protein is cleaved between the dyes, their distance increases, FRET decreases and the fluorescence of the first dye becomes measurable. Advantage: no separation of substrate and product is required, saving time.