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Lab session 2

Lab session 2. Determination of protein pI. IUG, 2012 TMZ. PRINCIPLE. Proteins are amphoteric electrolytes.

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Lab session 2

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  1. Lab session 2 Determination of protein pI IUG, 2012 TMZ

  2. PRINCIPLE • Proteins are amphoteric electrolytes. • At a pH value > pIof a proteininthe solution, the electrolysis of amino group- is inhibited while carboxyl- is electrolyzed. At this moment, the protein is the anion with negative charges. • On the contrary, at a pH value < pIof the protein, the electrolysis of carboxyl- is inhibited while amino- is electrolyzed. At this moment, the protein is the cation with positive charges.

  3. At pI of a protein.. • The number of positive charges is equal to that of negative charges in the protein molecules and the protein is the neutral ion. • At this moment , pH value in the solution is isoelectric point of the protein. • At pI the viscosity and the solubility of the protein are decreased.

  4. Experiment Reagents • Casein acetate sodium solution 1. Pure casein 0.25 g is placed into a beaker. 2. 20 ml distilled water and 5 ml 1 NNaOH are added into it. 3. After casein is dissolved completely,5 ml 1 N acetic acid is added into the beaker again. Then the solution is diluted to 50 ml with distilled water.

  5. Procedure 1. Five test tubes with the same diameter are numbered and the reagents are added into the tubes according to the following table.

  6. Volume in ml

  7. 30 min later at room temperature, the amount of precipitates is observed, which is represented by +, ++, +++, or ++++ , respectively. The pH value in the tube whose precipitates are the most is the isoelectric point of casein. (pI of casein is pH ~ 4.6). 3. Add 2 ml ethanol to each test tube, and make visual estimation of the solution clouding degree.

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