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Sunday, December 13, 2009

How to estimate proteolytic activity of pepsin Estimation of Protease Activity

Protease of proteolysis enzyme cafulyzes the breakdown of protein to peptides, and amine acids. There are 3 sources of proteases 1) Plant source 2) Animal source 3) Microbial source. Try sin, chemotropism, pepsin etc. are animal protease. They occur in gastro intestinal tract of all animals’ pepsin is an acid protease which has broad specificity. It hydrolyses peptide bonds formed by acidic aerobatic amino acids.
Photolytic activity of enzyme is determined by adding enzyme soln to a suitable under optimum conditions and estimating the products of hydrolysis by Ninhydrin colorimetric of pope-sternson method or Sarandon’s method.
In Sorenson titration, amino acid coln is treated with neutral HCHO soln and ethylene compound obtained is titrated with standard alkali. Following equation represents the equation of amino acid with HCHO.




Enzyme unit is defined as amount of amino acid liberated which will naturalize 1 ml of 0.1 NaOH under optimum condition after 2 hours of incubation of 370 c.

Reagents: - 3% pepsin, 0.1 NHCl, albumin 0.1 N NAOH.

Procedure: - Take 30 ml pepsin soln in a 250 ml conical flask Add 10 ml pf albumin and 10 ml of 0.1 NHCl. Immediately pipette out 10 ml of mixture and titrate against 0.1 N NaOH using phenol tholepins as indicator. Note the reading [A]. Add 20 ml of formaldehyde soln and continue the titration and note the reading [B]. Keep the remaining in the incubator for 2 hrs. Pipette out 10 ml of reaction mixture, and titrate against 0.1 N NaOH using phenol tholepins as indicator. Note the reading [A] and then add 20 ml of formaldehyde soln continue the titration. Note the reading [B]. Amount of free amino acid = [B’ – A’] – [B – A].

Observation:-
Solution in burette: 0.1N NaOH.
Solution in flask : Pepsin + albumin + 0.1 NHCI + HCHO
Indicator : Phenolphthalein.
End point : colorless to pink.

Burette reading:
Blank A = 1.8 ml
B = 2.5 ml
Sample A’= 1.8 ml
B’= 2.7 ml

Amount of free Amino acid = [B’- A’] – [B – A]
= [2.7 – 1.8] – [2.5-1.8]
= 0.2

Calculations:-
1 ml of 0.1N NaOH = 1unit
0.2 ml of 0.1N NaOH = 0.2 unit

10 ml of solution = 0.2 unit.
50 ml of Soln = 1 unit of pepsin.
therefore 30ml of pepsin = 1 unit of pepsin
therefore 100ml of pepsin = 3.33 units of pepsin

The protease activity is estimated to be 3.33 units
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