Dibasic sodium phosphate, 0.05 M— Dissolve 7.1 g of anhydrous dibasic sodium phosphate in water to make 1000 mL. Add 1 drop of toluene as a preservative.

Citric acid, 0.05 M— Dissolve 10.5 g of citric acid monohydrate in water to make 1000 mL. Add 1 drop of toluene as a preservative.

Casein substrate— Disperse 1 g of Hammersten-type casein in 50 mL of 0.05 M Dibasic sodium phosphate. Place in a boiling water bath for 30 minutes with occasional stirring. Cool to room temperature, and add 0.05 M Citric acid to adjust to a pH of 6.0 ± 0.1. Stir the solution rapidly and continuously during the addition of the 0.05 M Citric acid to prevent precipitation of the casein. Dilute with water to 100 mL. Prepare fresh daily.

Buffer solution (Phosphate-Cysteine Disodium ethylenediaminetetraacetate Buffer)—Dissolve 3.55 g of anhydrous dibasic sodium phosphate in 400 mL of water in a 500-mL volumetric flask. Add 7.0 g of disodium edetate and 3.05 g of cysteine hydrochloride monohydrate. Adjust with 1 N hydrochloric acid or 1 N sodium hydroxide to a pH of 6.0 ± 0.1, dilute with water to volume, and mix. Prepare fresh daily.

Trichloroacetic acid solution— Dissolve 30 g of reagent grade trichloroacetic acid in water, and dilute with water to 100 mL. This solution may be stored at room temperature.

Standard preparation— Weigh accurately 100 mg of USP Papain RS in a 100-mL volumetric flask, and add Buffer solution to dissolve. Dilute with Buffer solution to volume, and mix. Transfer 2.0 mL of this solution to a 50-mL volumetric flask, dilute with Buffer solution to volume, and mix. Use within 30 minutes after preparation.

Assay preparation— Transfer an accurately weighed amount of Papain, equivalent to about 100 mg of USP Papain RS, to a 100-mL volumetric flask, dilute with Buffer solution to volume, and mix. Transfer 2.0 mL of this solution to a 50-mL volumetric flask, dilute with Buffer solution to volume, and mix.

Procedure— Into each of 12 test tubes (18- × 150-mm) pipet 5.0 mL of Casein substrate. Place in a water bath at 40, and allow 10 minutes to reach bath temperature. Into each of two of the tubes (the tests are run in duplicate except for the blanks) labeled S1, pipet 1.0 mL of the Standard preparation and 1.0 mL of the Buffer solution, mix by swirling, note zero time, insert the stopper, and replace in the bath. Into each of 2 other tubes, labeled S2, pipet 1.5 mL of Standard preparation and 0.5 mL of Buffer solution, and proceed as before. Repeat this procedure for 2 tubes, labeled S3, to which 2.0 mL of Standard preparation is added, and for 2 tubes, labeled U2, to which 1.5 mL of Assay preparation and 0.5 mL of Buffer solution are added. After 60 minutes, accurately timed, add to all 12 tubes 3.0 mL of Trichloroacetic acid solution, and shake vigorously. With the 4 tubes to which no Standard preparation or Assay preparation were added, prepare blanks by pipeting, respectively, 1.0 mL of Standard preparation and 1.0 mL of Buffer solution; 1.5 mL of Standard preparation and 0.5 mL of Buffer solution; 2.0 mL of Standard preparation; and 1.5 mL of Assay preparation and 0.5 mL of Buffer solution. Replace all tubes in the 40 water bath, for 30 to 40 minutes, to allow to coagulate fully the precipitated protein. Filter through medium-porosity filter paper, discarding the first 3 mL of the filtrate (filtrates used are clear). Read the absorbances, at 280 nm, of the filtrates of all solutions against their respective blanks. Plot the absorbance readings for S1, S2, and S3 against the enzyme concentration of each corresponding level. By interpolation from this curve, taking into consideration dilution factors, calculate the potency in Units, in the weight of Papain taken by the formula: in which 50,000/3 is a factor derived by the expression 100(50/2)(10/1.5), C is the concentration, in mg per mL, obtained from the standard curve, and A is the activity of the Reference Standard in Units per mg.