AOAC Official Method 935.44 Papain Proteolytic Activity
Titrimetric Method
First Action 1935
A. Reagents
(a) Casein solution.—Make 6% solution of Hammersten casein (US Biochemical Corp, 26111 Miles Rd, Cleveland, OH 44128, USA) by rubbing 60 g with littleH2Oin mortar and gradually adding 60 mL 1M NaOH and H2O until volume totals 1 L. Heat viscous solution 30 min in boilingH2Obath, cool, and filter through glass wool if necessary.
(b) Citrate buffer solution.—Prepare 0.2M monosodium citrate solution by partial neutralization of citric acid with NaOH.
(c) Titrating solution.—Standardized 0.1M alcohol KOH.
(d) Indicator.—1% alcohol thymolphthalein solution.
B. Preparation of Test Sample
(a) Unactivated.—If enzyme preparation is solid, grind to smooth paste in small mortar with little freshly boiled, cold H2O. Suspend in cold boiledH2Oin proportion of 10 mg original preparation/mLH2O. After 5–10 min centrifuge suspension and discard sediment.
(b) Activated.—Proceed as in (a), but use half-saturated H2S-H2O instead of boiled H2O. After centrifuging, incubate enzyme solution 1 h at 40°C to complete activation.
