Study of Specific Activity of salivary Amylase


Activity of enzyme is defined as the micromoles of product formed per ml of enzyme solution under the optimum conditions. The determination of specific activity of enzyme involves two steps estimation of protein by F.C. Methos and estimation of product (glucose) by DNS method.

The FC method is one of the best methods for protein estimation. In this molybdate in the phosphomolybolate tungstate complex is reduced by tyrosine and tryptophan residues of proteins to give blue color.

 The formation of blue color is enhanced by cupric ions.

In DNS reaction, when alkaline solution of 3,5- Dinitro Salicylic (DNS) acid is heated with a reducing sugar, the acid is reduced to give a red color.

Reagents required:

  • 1% Starch
  • Alkaline copper reagent
  • Folin- Ciocalteau (FC) reagent
  • Standard protein solution

 Stock solution: Prepare 1mg/ml bovine serum albumin solutions

  •  Working Solution: Dilute 20 ml of stock solution to 100ml with distilled water.
  • Standard Glucose Solution: Prepare 1 mg/ml glucose solution
  • 3,5 Dinitro Salicylate Solution: Dissolve 1.0g of 3,5 Dinitro salicylic acid in 20 ml of 2N NaoH solution. Add this solution carefully to 50 ml of water containing 30g of Potassium sodium tartrate tetrahydrate, also known as Rochelle salt.  Make the volume to 100ml by distilled water.
  • Salivary amylase: Collect human saliva and dilute it 1:200 with phosphate buffer (pH 6.9)


Part A:

Estimation of protein in saliva by FC method:

Pipette aliquots of standard protein solution in the test tubes and make the final volume 1 ml with water in each case. Add 5ml of alkaline copper reagent to all the test tubes. After 10 minutes add 0.5ml of FC reagent. After 20 minutes, read the absorbance at 660nm against blank. Plot standard graph accordingly. In another fresh test tube take 1 ml of salivary amylase enzyme and add 5 ml of alkaline copper reagent. After 10 min, add 0.5ml FC reagent and after 20 minutes read the absorbance at 660 nm. Determine the amount of protein in the given salivary amylase solution by comparing the absorbance with standard curve.

 Part B:

 Estimation of glucose (product) by DNS method:

Pipette out different aliquots of standard glucose into different test tubes and make the final volume to 2.0ml with distilled water in each test tubes. Add 2ml of DNS reagent and mix it well. Keep the tubes in boiling water bath for 15 minutes. Cool it and mix the content thoroughly and read the absorbance at 540 nm against the blank solution. Plot the standard graph.

In another test tube take 1 ml of salivary amylase enzyme and 1ml of 1% starch.  After 10 minutes determine the amount of product-glucose formed by adding 2ml of DNS reagent and boil it for 15minutes accordingly and read the absorbance at 540nm and compare it with standard graph.

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