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

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

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

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

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

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

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

Procedure— Into each of 12test tubes (18-×150-mm)pipet 5.0mLof Casein substrate.Place in a water bath at 40,and allow 10minutes to reach bath temperature.Into each of two of the tubes (the tests are run in duplicate except for the blanks)labeled S1,pipet 1.0mLof the Standard preparationand 1.0mLof the Buffer solution,mix by swirling,note zero time,insert the stopper,and replace in the bath.Into each of 2other tubes,labeled S2,pipet 1.5mLof Standard preparationand 0.5mLof Buffer solution,and proceed as before.Repeat this procedure for 2tubes,labeled S3,to which 2.0mLof Standard preparationis added,and for 2tubes,labeled U2,to which 1.5mLof Assay preparationand 0.5mLof Buffer solutionare added.After 60minutes,accurately timed,add to all 12tubes 3.0mLof Trichloroacetic acid solution,and shake vigorously.With the 4tubes to which no Standard preparationor Assay preparationwere added,prepare blanks by pipeting,respectively,1.0mLof Standard preparationand 1.0mLof Buffer solution;1.5mLof Standard preparationand 0.5mLof Buffer solution;2.0mLof Standard preparation;and 1.5mLof Assay preparationand 0.5mLof Buffer solution.Replace all tubes in the 40water bath,for 30to 40minutes,to allow to coagulate fully the precipitated protein.Filter through medium-porosity filter paper,discarding the first 3mLof the filtrate (filtrates used are clear).Read the absorbances,at 280nm,of the filtrates of all solutions against their respective blanks.Plot the absorbance readings for S1,S2,and S3against 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 50,000/3is a factor derived by the expression 100(50/2)(10/1.5),Cis the concentration,in mg per mL,obtained from the standard curve,and Ais the activity of the Reference Standard in Units per mg.