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Papain
Definition
Papain is a proteolytic enzyme preparation derived from fruits of
papaya. It may contain lactose or dextrin.
Enzyme Activity
The enzyme activity of Papain is not less than 300,000 units per
gram.
Description
Papain occurs as whit to light yellow-brown powders. It is odorless or
has slight characteristic odors.
Identification
(1) To 10 ml of a liquid including 20 % of powdered skim milk,
adjusted to pH 5.5 with diluted acetic acid (3 → 50), add 0.01 g of Papain, and warm to
37℃. The liquid coagulates.
(2) The solution of Papain(1 → 500) exhibits absorption maximum at a wavelength
of 270 − 280 nm.
Purity
(1)
Heavy metals
Not more than 40 µg/g as Pb (0.50 g, Method 2,
Control solution Lead Standard Solution 2.0 ml).
Not more than 10 µg/g as Pb (1.0 g, Method 1).
(2)
Lead
(3)
Arsenic
Not more than 4.0 µg/g as As2O3 (0.50 g, Method 3, Apparatus B).
Microbial Limits Proceed as directed under the Microbial Limit Tests. The total
viable aerobic count is not more than 50,000/g and no Escherichia Coli is observed.
Enzyme Activity Determination
(i)
Sample Solution
Dissolve 8.75 g of L-cystein hydrochloride in about 800 ml of water, add 2.23 g of
disodium ethylenediaminetetraacetate, and dissolve. Adjust the pH to 4.5 with 1 mol/l
sodium hydroxide TS and add water to make 1,000 ml. Use this solution as the diluent.
Weigh accurately about 0.50 g of Papain, dissolve in the diluent, and make exactly
100 ml. Measure exactly 1 ml of the solution, and add the diluent to make exactly 50 ml.
Centrifuge this solution, if necessary. Dilute the supernatant liquid with the diluent to
prepare a solution containing 20 to 100 units per ml.
(ii) Procedure
Measure exactly 5 ml of casein TS (pH 8.0), transfer into a test tube, and warm for 5
minutes at 37±0.5℃. Add 1 ml of the sample solution, shake immediately, and react for
10 minutes at 37±0.5℃. Add 5 ml of trichloroacetic acid TS, shake, allow to stand for 30
minutes at 37±0.5℃, and filter through a filter paper for quantitative analysis (5C).
Discard the first 3 ml of the filtrate, measure the absorbance (At) of the subsequent
filtrate at 275 nm using water as the reference.
Measure exactly 1 ml of the sample solution, add 5 ml of trichloroacetic acid TS,
and shake well. Add 5 ml of casein TS (pH 8.0), shake well, allow to stand for 30
minutes at 37±0.5℃.
Measure the absorbance (Ab) of this solution, proceeding in the same manner as for the
measurement of absorbance At. Separately, measure the absorbances (AS and AS0) of
Tyrosine Standard Solution and 0.1 mol/l hydrochloric acid at 275 nm using water as
the reference. Calculate the enzyme activity by the formula given below. One unit of the
enzyme activity is the quantity of enzyme which increases an absorbance equivalent to
1 µg of tyrosin per minute when the test is performed under this procedure.
The Enzyme Activity of Papain (units/g) =
where
(A t − A b ) × 50 11 1,000
,
×
×
A s − A so
10
W
W ＝ weight (mg) of Papain in 1 ml of the sample solution.