Custom Search

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
========================================================================
This website is a guide for for pharmacy students , pharmaceutical professional working in pharmaceutical regulatory affairs , pharmaceutical manufacturing , quality assurance , pharmaceutical formulation development , research and development.
This week we are publishing a series of articles on biochemistry, over this website , for our pharma students world wide .
We recomend you to sebscribe to this website and receive our articles directly by email when ever we update this website.

This website is most favorite Amongst proffesionals in almost all pharmaceutical companies in USA , Pharmaceutical companies in UK , Pharmaceutical companies in Japan , Canada , UAE , Pharmaceutical companies in India 

Following are some of articles which;will be useful for you in further understanding of aspects of sterile dosage form manufacturing

Pharmaceutical Validation

Cleen Room Classification

Pharmaceutical Industry

Pharmaceutical Manufacturing

Clinical Trials

Media fill

Regulatory Affairs in Pharmaceutical Industry

No comments:

Posted by: Martin

How to sucessfuly activate and receive our articles on pharmaceuticals by E-Mail

Dear readers if you wish to receive our articles on pharmaceutical automatically into your email Inbox , as and when we write a new article over this website, then follow these 3 simple steps.


1.You should write your Email ID in the box provided and push the subscribe butten


2.Enter the text code that we show you in a small new browser window after you enter your email ID.

3.Log in to your email and Cheke our verification email sent by "FEEDBURNER" , there you should verify the link we send you by email.




Enter your email address get our articles by email whenever this website is updated





Delivered by FeedBurner



Join the list of our readers from Universities ,Research and Development centers Pharmaceutical Companies from all over the world.











Pharma Research Regulatory Guidelines Website
Texas Dallas, TX 75211 US
Email: editor@pharmacistspharmajournal.org Website: http://pharmacistspharmajournal.org/
Copyright © 2008-2015 all rights reserve www.pharmacistspharmajournal.org