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  Section: Plant Lab Protocols
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Methodology for Enzymes

(Papainase EC
Papain, one of the plant proteases, is present in the papaya latex. Though the latex contains other proteases also, papain is the most studied one because of its abundance. It is largely used in brewing and food industries. It is also used in cosmetics and pharmaceutical industries.
An artificial substrate Benzoyl, L-Arginine p-nitroanilide (BAPNA) is hydrofysed by papain and the color intensity of the released p-nitroaniline is measured at 410nm. With the molar extinction coefficient of p-nitroaniline, the activity of the enzyme is calculated. Alternatively the color intensity of the released p-nitroaniline in the sample may be compared with the standard p-nitroaniline.

Tris-HCl buffer 50mM (pH 7.5): Dissolve 605mg Tris in 50mL of distilled water. Adjust to pH 7.5 with 0.05N hydrochloric acid and make up volume to 100mL.
To the above buffer (100mL) add 87.8mg of cysteine hydrochloride (0.005M) and 74.4mg of EDTA (0.002M) and dissolve completely.
Buffered Substrate Solution
   Dissolve 43.5mg BAPNA in 1mL of dimethyl sulphoxide and make up the volume to 100mL with Tris-HCl buffer containing 5mM cysteine and 2mM Ethylenediamine tetra acetate.
Acetic Acid 30%
Enzyme Source
   Dissolve papain to a concentration of 0.1mg/mL distilled water. (For crude latex 100mg/mL will give measurable level of papain.)
Pipette out 0.5mL of papain solution into a test tube. (May be carried out in duplicate with different aliquots of enzyme).
Make up the volume to 1.0mL with Tris-HCl buffer.
Add 5mL of substrate solution.
Incubate for 25min at 25°C.
Terminate the enzyme reaction by adding 1mL of 30% acetic acid.
Measure the absorbance of the released p-nitroaniline at 410nm against a control (without the enzyme) in a spectrophotometer.
Molar extinction coefficient of p-nitroaniline =

  = 8,800/lmole/cm.
1 millimole/L =
= 8.8A
1 m mole/L =
= 0.0088A
1 m mole/mL = 8.8
If the absorbance is 8.8, amount of p-nitroaniline
                                = 1 m mole/mL
If X is the absorbance of the sample,
1 m mole/mL
x X
= X
1 m mole/mL
This is for one mL of the sample for 25min.
For 7mL (volume of the assay mixture) of the sample solution
X m mole
The assay mixture contains 0.5mL of the enzyme
Therefore, Activity of the enzyme in 1mL
X m mole x 7.0
8.8 x 0.5 x 25
m mole of p-nitroaniline released per min)
From this calculate the activity per g sample. For specific activity, determine the protein content of the enzyme solution by Lowry's method (See section on protein) and express per mg protein.

1.  If p-nitroaniline is available, a standard graph may be drawn and the quantity of p-nitroaniline released by the enzyme can then be calculated.
2.  Papain can also be estimated using casein, albumin or Benzoylarginine ethylester as substrate where the unit of enzyme activity will differ.   But for comparison of papain activity among varieties, any one of the substrates may be used.
1.  Arnon, R (1970) In: Methods Enzymol 19 (Eds Perlmann Lorand, L) Academic Press New York p 228.

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