Hi Hassan, this will be easy. Choose a specific chromogenic substrate for each enzyme.

Measure the activity of each enzyme in a spectrophotometer by change in absorbance and measure only the linear part of the enzyme activity per time. This will give you dA/dt value. Divide that by the protein concentration used in the assay will give you specific activity.

The more the enzymes are purified, the sharper the incline of the linear part of the enzyme assay will become with less protein used. Divide that dA/dt by the protein concentrations used in the assay will tell you if the specific activity is increasing.

Specific Activity = Enzyme (unit) Activity/ protein concentration

The unit is usually U/mg protein.

Where U = Enzyme unit

Dear Adeyemi,

Very thanks for your reply...U can be calculated from standard graph or it is the reading(absorbance) that I have got from uv-spectrophotometer??? Can you please provide me with any research article mentioning this formula???

Waiting for your response

For those following:

formula for calculating enzyme activity (U) with respect to the standard used.

For amylase, you will have to plot a glucose standard graph of Absorbance against concentration.

Then use this following formulae to calculate the Enzyme Activity (U)

Enzyme Act = Absorbance of sample/(Slope of standard graph X volume of sample used X Time of incubation for reaction)

The same goes for other enzymes too with respect to their standards

ACID PHOSPHATASE (E.C. 3.1.3.2)

(Schmidt, 1955)

Principle: Phosphatases liberated inorganic phosphorus from organic phosphorus esters. Acid phosphatase hydrolyzes a number of phosphomonoesters and phosphoproteins. Phosphodiesters are not hydrolysed by either of them.

Acid phosphatases can be assayed by measuring the amount of inorganic phosphorus released from sodium glycerophosphate. Phosphorus is estimated by Fiske-Subbarow (1925) method.

Reagents:

· Acetate buffer : 0.1 M pH 5.0

Stock A: 0.2 N acetic acid 5.68 ml CH3 COOH 500 ml-1

Stock B: 0.2 M Sodium acetate 13.6 g C2 H3 O2 Na.3 H2O 500 ml-1. Mix stock A 148 ml and stock B 352 ml and diluted to 1L with GDW

· M Na-β glycerophosphate = 0.315 g/10 ml GDW

· 10% Tri chloro acetic acid (TCA)

· Ammonium molybdate: Ammonium Molybdate Reagent:

Dissolve 12.5 g ammonium molybdate in 200 ml G.D.W. or more. Then add 150 ml of 10 N H2SO4 and make the volume 500 ml by GDW (Fiske and Subbarow, 1925 ).

· 1,2,4 Amino Napthal Sulphonic Acid:

250 mg 1,2,4 amino napthal sulphonic acid dissolved by grinding in 13.7% sodium meta sulphide Na2S2O5 (upto 97.5 ml). Add 0.5 g sodium sulphite make the volume to 100 ml. Filter and store in a coloured glass stoppered bottle in fridge

Procedure:

Take 500 mg of plant material grind in child pestle and mortar in10 ml GDW

Acetate buffer pH 5.0 = 0.5 ml

+

Enzyme extract = 0.4 ml

+

Na-β glycerophosphate = 0.1 ml

†

Stand for 20 minutes at 30o C

†

Add TCA 10% = 1.0 ml

†

In corresponding blanks TCA should be added prior to addition of Na-β glycerophosphate (to stop the reaction)

†

Centrifuge at 0o C

†

Draw aliquot from supernatant of samples and blanks for the estimation of P (Fiske and Subbarow, 1925) described as below-

Take supernatant in both blank and sample = 1.0 ml GDW = 7.6 ml

ammonium molybdate reagent = 1.0 ml

1, 2, 4, amino napthol sulphonic acid. = 0.4 ml

†

Stand for 10 minutes

Read absorbance at 640 nm. (red filter) within ½ hour in a spectrophotometer.

From standard graph read the μg phosphorus.

Unit: Inorganic phosphorus (Pi) liberated 100 mg-1 fresh weight or mg-1 protein

Thanks Dr.Girish for your kind response...