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Official Method Titrimetric Methods

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AOAC Official Method 935.44 Papain Proteolytic Activity Titrimetric Method First Action 1935.rar,Official,Method,Titrimetric,Methods

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47.5.01 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)byrubbing60gwithlittleH2Oinmortarandgraduallyadding 60mL1MNaOHandH2Ountilvolumetotals1L.Heatviscousso- lution30mininboilingH2Obath,cool,andfilterthroughglasswool 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.Ifenzymepreparationissolid,grindtosmooth paste in small mortar with little freshly boiled, cold H2O. Suspend in coldboiledH2Oinproportionof10mgoriginalpreparation/mLH2O. After 510 min centrifuge suspension and discard sediment. (b) Activated.Proceed as in (a), but use half-saturated H2SH2O instead of boiled H2O. After centrifuging, incubate en- zyme solution 1 h at 40C to complete activation. C. Determination Place10mLcaseinsolutionandsmallchargeof4mmdiameterglass beadsineachofseveral125mLglass-stopperedbottles,andbringbot- tlesandcontentsto40C.Adddesiredvolumeofpreparedenzymeso- lution,but4mL.Ifthisamountisinsufficient(see935.44D),prepare moreconcentratedsolutionofenzyme.Immediatelyaddexactly3mL buffer solution (pH of system should be 5.0 0.1). Vigorously shake bottle few s and place in constant temperature H2O bath at 40C. Incubate 20 min at 40C, counting time from addition of buffer. Add 1 mL indicator and begin titrating with alcohol KOH solution. As soon as deep blue appears, shake bottle until color is discharged orprecipitateiscompletelydissolved.(Itisusuallybesttoaddalkali inca0.5mLportions.)Whenallprecipitatedcaseindissolves,trans- fersolutionto400500mLflaskandrinsebottle2or3timeswithal- cohol, using total of 25 mL. Add enough KOH solution to restore blue;thenadd175mLboilingalcohol.CarefullyaddmoreKOHso- lution until pale but distinct blue persists in solution. Makecontroltitrationexactlyasdescribed,butdoitimmediately afteradditionofbuffer,withoutanyincubationtime.Differencebe- tween titration of undigested test solution and that of digested test solution is measure of proteolytic activity of enzyme. D. Calculation of Proteinase Unit Forsmalleramountsofenzyme,extentofhydrolysisdeterminedby abovetitrationisstraightlinefunctionofamountsofpapainused.For accurate work, determine this straight line by making several titra- tionswithdifferentamountsofenzyme.Ifamountsofpapainusedare too large, straight-line relationship no longer holds; if they are too small, determination is inaccurate. Amounts of enzymes giving titra- tion differences of 0.61.2 mL 0.1M KOH are recommended. Unit of papain is amount of enzyme that produces, under condi- tions outlined, titration difference of 1 mL 0.1M KOH, determined either graphically or arithmetically. Value of original preparation is thenexpressedinunits/mg,orasmg