Papain

Papain [9001-73-4]

  Papain is a purified proteolytic substance derived from Carica papaya Linné(Fam.caricaceae).papain,when assayed directed herein, contains not less than 6000 units per mg. Papain of a higher digestive power may be reduced to the official standard by admixture with papain of lower activity ,lactose,or other suitable diluents.

  One USP Unit of papain is the activity that releases the of 1μg of tyrosine from a specified casein substance under the conditions of the Assay， using the enzyme concentration that liberates 40μg of tyrosine per mL of test solution.

Packaging and storage－Preserve in tight,light-resistant containers in a cool place.

USP reference standards (11)—USP papain RS.

pH<791>:between 4.8 and 6.2 in a solution (1 in 50).

Loss on drying <731>—Dry it in a vacuum oven at 60℃ for 4 hours : it losses not more than 7.0% of its weight.

Assay (casein digestive power)—

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

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

   Casein substrate—Disperse 1 g of Hammersten-type casein in 50 mL on 0.05M 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 0.05M 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 3.55g of anhydrous dibasic phosphate in 400mL of water in a 500-mL volumetric flask.Add 7.0 g of disodium EDTA 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 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 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 100mg of USP Papain RS, to a 10-mL volumetric flask, dilute with Buffer solution to volume, and mix. Transfer 2.0 mL of this solution to a 50-mL volumetric flsk, 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 1.0mL 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 1.5mL of standard . preparation and 0.5 mL of Buffer solution , and proceed as before . Repeat this procedure for 2 tubes ,labeled S3to which 2.0mL of standard preparation is added , and for 2 tube ,labled U2,to which 1.5mL of Assay preparation and 0.5mL  Buffer solution are added.After 60 minutes,accurary 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 ,1.0mL of standard preparation and 1.0 mL of Buffer solution 1.5mL of standard preparation and 0.5 mL of Buffer solution;2.0mL of standard preparation;and 1.5mL 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 if 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.:

                (50,000/3)CA,

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. 

翻译仅供参考：

木瓜蛋白酶

木瓜蛋白酶[9001-73-4]

木瓜蛋白酶是一种源自番木瓜乳汁（Fam.caricaceae）的纯化的水解蛋白物。木瓜蛋白酶在这里定向检测时其含量不得少于6000单位每毫克。具有较高的消化能力的木瓜蛋白酶也许可以通过掺加低活力的木瓜蛋白酶，乳糖或者其他合适的填充物来降低消化能力以达到官方的标准。

定义木瓜蛋白酶一个USP活力单位为在测定条件下指定的酪蛋白物质释放1μg酪氨酸所需要的量，所用的酶试液的浓度相当与每毫升释放40μg酪氨酸 。

包装与保藏—包装要包紧，保存在避光，阴凉的地方        

USP 参考标准（11）—USP 木瓜蛋白酶 R.S

pH<791>在溶液中介于4.8与6.2之间（1 in 50？）

干燥失重<731>—60℃时在真空干燥箱中干燥4小时：失重不得少于本身的7.0％

检测（酪蛋白消化能力）

    0.05M磷酸氢二钠溶液—准确称取7.1g无水磷酸氢二钠用蒸馏水水溶解并定容至1000mL。加一滴甲苯作为防腐剂。

    0.05M柠檬酸—准确称取10.5g柠檬酸一水合物用蒸馏水溶解并定容至1000mL。加一滴甲苯作为防腐剂。酪蛋白底物—将1g Hammersten-type 酪蛋白分散于50mL磷酸氢二钠溶液中，将上述溶液沸水浴加热30分钟并在必要时搅拌。酪蛋白溶解后冷却至室温再用0.05M柠檬酸调节pH至6.0±0.1。为了防止酪蛋白沉淀，加入0.05M柠檬酸的过程中要不断的快速搅拌。然后将上述溶液定容至100mL，临时现配。

缓冲溶液（磷酸盐—半胱氨酸－磷酸氢二钠EDTA－2Na缓冲）

——准确称取3.55g二代磷酸盐用400mL水溶解移入到500mL的容量瓶中，同时加入7.0g EDTA及3.05g 半胱氨酸盐酸盐一水物。用1mol/L盐酸或1mol/L氢氧化钠溶液调至pH6.0±0.1。用水稀释至刻度，混匀，临用现配。

三氯乙酸溶液—准确称取30g 试剂级三氯乙酸用水溶解并定容到100mL。此溶液可以保存于室温。

标准试剂—精确称取100mg RS级USP木瓜蛋白酶置于100mL容量瓶中，加入缓冲溶液使之溶解，再用缓冲溶液定容至刻度，混匀。移取2.0mL此溶液置于50mL容量瓶中，然后用缓冲溶液定容至刻度，混匀。配好后30min内用。

待测试剂—将准确称取的与100mg USP RS级木瓜蛋白酶当量的木瓜蛋白酶移入100mL的容量瓶中，用缓冲溶液稀释至刻度，混匀。移取2mL此溶液到50mL容量瓶中并用缓冲溶液定容至刻度，混匀。

步骤——用吸量管每次吸取5ml酪蛋白底物分别加往12支试管（18×150mm）中，将试管置于40°水浴加热10分钟以使溶液达到水浴温度。然后每两管（除空白之外其他管操作完全相同）标上S1 ，往上述两管中吸入1.0mL标准试剂和1.0mL缓冲溶液，涡流混匀，记下开始时间，加入终止剂，放回水浴中。其他两管标上S2 吸入1.5mL标准溶液和0.5mL缓冲溶液，以后操作同前。对两试管重复此过程标上S3 吸入2.0mL标准试剂，对后拿两试管标上U2 加入1.5mL待测试剂和0.5mL缓冲溶液。精确计时60分钟后，往所有的12支试管中加入3.0ml三氯乙酸溶液并剧烈震荡。同时准备四支没有加入标准试剂或待测试剂的试管作为空白对照分别加入1.0mL标准试剂和1.0mL缓冲溶液，1.5mL标准试剂和0.5mL缓冲溶液，2.0mL标准试剂以及1.5mL待测试剂和0.5mL缓冲溶液。将所有试管放回40℃水浴30分钟到40分钟以便使蛋白质沉淀物完全凝结。用中等孔径滤纸过滤，弃去首先收集的3mL滤液（所用的滤液要是清澈的）。分别读出溶液滤液在其相应的空白作为参比溶液下在280nm处的吸光度。将S1,S2­ 和 S3对应的吸光度读数对各自相应的酶浓度水平作图。通过对图像用内插法，并考虑稀释因子，用单位，木瓜蛋白酶质量计算通过下述公式计算其效价：(50,000/3)CA

   其中50,000/3源自表达式100（50/2）（10/1.5）得到的因数，C表示浓度

   单位：mg/mL，从标准曲线获得，A表示参比标准的活力单位：单位/毫克