I followed some literature concerning the protease assay but I am a little bit confused about what substrate should be taken for the assay. Yes, casein is cheaper than azocasein, but is there any differences among them. Both are universally accepted or not? Thanks in advance for your replies.
As I understand it, azocasein is casein modified with a dye, allowing a simple continuous, colorimetric assay for proteolysis (absorbance at 440 nm), rather than an endpoint assay. Thus, using azocasein should allow more data to be collected in the same amount of time, especially if one is using a plate reader and multiwell assay plates.
Hi Abhas, just to add to Katarzyna's answer, both casein and azocasein are acceptable assays for measuring proteinases activities.
The only advantage that azocasein has is that its degradation products are easier to measure by regular spectrophotometers.
If you can measure casein degradation products by UV-Vis spec, then that will be good enough for publication.
Thank you all for valuable suggestions.
Dr. Katarzyna Styła, can you send me the full text of the publication you mentioned in the answer??
both are accepted..caseinolytic you check at 660nm and Azocasein degradation at 440nm..
and you can also study action of your protease(s) on casein, azocasein and hemoglobin.. that will add to your data and interpretations on substrate specificity while characterizing the protein..
Casein is a good substrate for a preliminary study. see
Nedra El Hadj-Ali, Rym Agrebi, Basma Ghorbel-Frikha, Alya Sellami-Kamoun, Safia Kanoun and Moncef Nasri (2007), Biochemical and molecular characterization of a detergent stable alkaline serine-protease from a newly isolated Bacillus licheniformis NH1. Enzyme and Microbial Technology, 40, Issue 4: 515-523
Kembhavi, A.A., Kulkarni, A. and Pant, A.A. (1993) Salt-Tolerant and Thermostable Alkaline Protease from Bacillus subtilis NCIM No 64. Applied Biochemistry and Biotechnology, 38, 83-92.
You can use Casein or Azocasein, the methods are similar, same principle: You measure the ammount of low molecular weight molecules, released from sustrate (casein or azocasein) that are not precipitated by TCA. Supernatant in recovered by centrifugation and then you measure absorbance: Casein method, at 280nm using a UV cuvettes. Azocasein method, at 440 nm, in any type of cuvettes. But there is a difference about what is measured. In casein method you measure free aminoacids (aromatic ones) that absorbe al UV region (275-280 nm, Tyrosine and Tryptophane, and 267 Phenylalanine), and also peptides containing them. In contrat in Azocasein method you measure any free aminoácid or peptide contaning the Azo dye. It means that in the first one you measure aminoacids, and in the second one you measure the dye.
Both methods are end point, well acepted, you use the better for your interest.
A tyrosine standard curve is used, in the Casein Method, to calculate activity in terms of tyrosine equivalente released by time.
In Azocasein method, usually activity is expressed in terms of Aborsorbance increase at 440 nm by time.
HN
OK, apparently I was wrong about azocasein allowing for a continuous readout. There are examples of continuous protease assays, however. Here is one that uses fluorescence:
http://tools.lifetechnologies.com/content/sfs/manuals/mp22132.pdf
As mentioned by others both casein and azocasein are accepted substrate for protease assay depending on the time most suitable to you and spectrophotometer type available in your lab. It is quite important to define the unit enzyme activity in whatever method/substrate you used. Usually 1 international unit for protease activity equals to 1 micromole tyrosine released per minute. As there are various protease of interest such as keratinase, collagenase, fibrin degrading protease and others, I usually use casein and other specific substrates for a particular protease. For example, when I want to analyze my fibrin degrading protease, then I use casein and also fibrin and fibrinogen as substrates in the enzyme assay. Similarly in my collagenase research I used casein and collagen as my substrate.
Thanx to all for their valuable suggestions.
And Wish all a very very HAPPY NEW YEAR.... :-)
If activity of protease using casein as a substrate is measured at 280nm. Then why is it in the preparation of tryrosine standard curve we have to measure at 660nm?
Anybody knows the calculation of protease activity ..when i used Azocasein or casein as a substrate?
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