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. 

翻譯僅供參考：

木瓜蛋白酶

木瓜蛋白酶[9001-73-4]

木瓜蛋白酶是一種源自番木瓜乳汁（Fam.caricaceae）的純化的水解蛋白物。木瓜蛋白酶在這里定向檢測時(shí)其含量不得少于6000單位每毫克。具有較高的消化能力的木瓜蛋白酶也許可以通過摻加低活力的木瓜蛋白酶，乳糖或者其他合適的填充物來降低消化能力以達(dá)到官方的標(biāo)準(zhǔn)。

定義木瓜蛋白酶一個(gè)USP活力單位為在測定條件下指定的酪蛋白物質(zhì)釋放1μg酪氨酸所需要的量，所用的酶試液的濃度相當(dāng)與每毫升釋放40μg酪氨酸 。

包裝與保藏—包裝要包緊，保存在避光，陰涼的地方        

USP 參考標(biāo)準(zhǔn)（11）—USP 木瓜蛋白酶 R.S

pH<791>在溶液中介于4.8與6.2之間（1 in 50？）

干燥失重<731>—60℃時(shí)在真空干燥箱中干燥4小時(shí)：失重不得少于本身的7.0％

檢測（酪蛋白消化能力）

    0.05M磷酸氫二鈉溶液—準(zhǔn)確稱取7.1g無水磷酸氫二鈉用蒸餾水水溶解并定容至1000mL。加一滴甲苯作為防腐劑。

    0.05M檸檬酸—準(zhǔn)確稱取10.5g檸檬酸一水合物用蒸餾水溶解并定容至1000mL。加一滴甲苯作為防腐劑。酪蛋白底物—將1g Hammersten-type 酪蛋白分散于50mL磷酸氫二鈉溶液中，將上述溶液沸水浴加熱30分鐘并在必要時(shí)攪拌。酪蛋白溶解后冷卻至室溫再用0.05M檸檬酸調(diào)節(jié)pH至6.0±0.1。為了防止酪蛋白沉淀，加入0.05M檸檬酸的過程中要不斷的快速攪拌。然后將上述溶液定容至100mL，臨時(shí)現(xiàn)配。

緩沖溶液（磷酸鹽—半胱氨酸－磷酸氫二鈉EDTA－2Na緩沖）

——準(zhǔn)確稱取3.55g二代磷酸鹽用400mL水溶解移入到500mL的容量瓶中，同時(shí)加入7.0g EDTA及3.05g 半胱氨酸鹽酸鹽一水物。用1mol/L鹽酸或1mol/L氫氧化鈉溶液調(diào)至pH6.0±0.1。用水稀釋至刻度，混勻，臨用現(xiàn)配。

三氯乙酸溶液—準(zhǔn)確稱取30g 試劑級三氯乙酸用水溶解并定容到100mL。此溶液可以保存于室溫。

標(biāo)準(zhǔn)試劑—精確稱取100mg RS級USP木瓜蛋白酶置于100mL容量瓶中，加入緩沖溶液使之溶解，再用緩沖溶液定容至刻度，混勻。移取2.0mL此溶液置于50mL容量瓶中，然后用緩沖溶液定容至刻度，混勻。配好后30min內(nèi)用。

待測試劑—將準(zhǔn)確稱取的與100mg USP RS級木瓜蛋白酶當(dāng)量的木瓜蛋白酶移入100mL的容量瓶中，用緩沖溶液稀釋至刻度，混勻。移取2mL此溶液到50mL容量瓶中并用緩沖溶液定容至刻度，混勻。

步驟——用吸量管每次吸取5ml酪蛋白底物分別加往12支試管（18×150mm）中，將試管置于40°水浴加熱10分鐘以使溶液達(dá)到水浴溫度。然后每兩管（除空白之外其他管操作完全相同）標(biāo)上S1 ，往上述兩管中吸入1.0mL標(biāo)準(zhǔn)試劑和1.0mL緩沖溶液，渦流混勻，記下開始時(shí)間，加入終止劑，放回水浴中。其他兩管標(biāo)上S2 吸入1.5mL標(biāo)準(zhǔn)溶液和0.5mL緩沖溶液，以后操作同前。對兩試管重復(fù)此過程標(biāo)上S3 吸入2.0mL標(biāo)準(zhǔn)試劑，對后拿兩試管標(biāo)上U2 加入1.5mL待測試劑和0.5mL緩沖溶液。精確計(jì)時(shí)60分鐘后，往所有的12支試管中加入3.0ml三氯乙酸溶液并劇烈震蕩。同時(shí)準(zhǔn)備四支沒有加入標(biāo)準(zhǔn)試劑或待測試劑的試管作為空白對照分別加入1.0mL標(biāo)準(zhǔn)試劑和1.0mL緩沖溶液，1.5mL標(biāo)準(zhǔn)試劑和0.5mL緩沖溶液，2.0mL標(biāo)準(zhǔn)試劑以及1.5mL待測試劑和0.5mL緩沖溶液。將所有試管放回40℃水浴30分鐘到40分鐘以便使蛋白質(zhì)沉淀物完全凝結(jié)。用中等孔徑濾紙過濾，棄去首先收集的3mL濾液（所用的濾液要是清澈的）。分別讀出溶液濾液在其相應(yīng)的空白作為參比溶液下在280nm處的吸光度。將S1,S2­ 和 S3對應(yīng)的吸光度讀數(shù)對各自相應(yīng)的酶濃度水平作圖。通過對圖像用內(nèi)插法，并考慮稀釋因子，用單位，木瓜蛋白酶質(zhì)量計(jì)算通過下述公式計(jì)算其效價(jià)：(50,000/3)CA

   其中50,000/3源自表達(dá)式100（50/2）（10/1.5）得到的因數(shù)，C表示濃度

   單位：mg/mL，從標(biāo)準(zhǔn)曲線獲得，A表示參比標(biāo)準(zhǔn)的活力單位：單位/毫克