CN1057608C - 一种亲和层析材料及制备方法和应用 - Google Patents

一种亲和层析材料及制备方法和应用 Download PDF

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CN1057608C
CN1057608C CN95114253A CN95114253A CN1057608C CN 1057608 C CN1057608 C CN 1057608C CN 95114253 A CN95114253 A CN 95114253A CN 95114253 A CN95114253 A CN 95114253A CN 1057608 C CN1057608 C CN 1057608C
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inhibitor
arrowhead
affinity chromatographic
chromatographic material
kallikrein
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CN1141212A (zh
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傅和亮
谢永立
何栏
巫锦娣
郑少亮
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Guangdong Tianpu Biochemical Medicine Co., Ltd.
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TIANPU BIOCHEMICAL PHARMACEUTICAL CO Ltd GUANGDONG
Bopu Biological Tech Co Ltd Guangzhou City
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Abstract

一种亲和层析材料，满足下列结构要求R-茨菇抑制剂，特定载体R采用不溶于水的基质材料，如各类琼脂糖，在一定条件下与茨菇抑制剂交联，制备一种亲和层析材料，从而利用该层析材料去除蛋白质中蛋白酶、激肽释放酶。

Description

一种亲和层析材料及制备方法和应用
本发明涉及生物化学领域。
茨菇抑制剂为新鲜茨茹中提取的两种多功能结晶蛋白酶抑制剂A和B的混合物，这两种蛋白酶抑制活性上略有不同，例如抑制剂A对糜蛋白酶和激肽释放酶亲和性较强而抑制B对胰白酶亲和性较强。可见，怎样利用茨茹抑制剂更好地去除蛋白质中蛋白酶和激肽释放酶，是目前该领域仍需解决的课题。
本发明的目的在于提供一种茨茹抑制剂一载体制备的亲和层析材料该层析材料的制备方法，以及采用该层折材料去除蛋白质中蛋白酶、激肽释放酶的方法。
本发明满足下列结构要求：
R＿茨菇抑制剂
这里R代表一种不溶于水的基质材料，包括各种类型的琼糖。
本发明中亲和材料的制备如下：
1、茨茹抑制剂的制备：
取新鲜茨菇块茎，去除外皮洗净、切碎、加少许去离子水匀浆，用200目滤布过滤，残渣用少量去离子水搅拌，浸泡过滤，和并滤液，调节PH至3.8～4.0。弃去沉淀，取上清加硫酸铵至饱和度为50％，沉淀蛋白、质弃上清，沉淀加水溶解用去离子水透析除盐，完毕后置65～75℃水浴中热变性5分钟，离心去除沉淀，上清调PH至7.0，用0.015M磷酸缓冲溶液透析。
取二乙基氨基乙基纤维素(DEAE-cellulose)凝胶装栓，先用0.015mol/L(PH7.8)磷酸缓冲溶液平衡，将上述溶液从柱上端加入，用0.05mol/L(PH7.8)磷酸缓冲溶液冲洗柱床，然后用0.012mol/L(PH7.8)磷酸缓冲溶液洗脱，收集蛋白质抑制剂洗脱峰，将洗脱峰用饱和硫酸铵透铵透析至沉淀不再增加，离心收集沉淀。加水溶解，用去离子水透析除盐，冷冻干燥后即得。
2、茨菇抑制剂与载体交联：
任何适合的不溶于水的基质材料均可作为载体材料，例如：琼脂糖凝胶(包括：琼脂糖2B、琼脂糖4B、琼脂糖6B和二乙基氨基琼脂糖)，将载体事先用溴化氰或其他激活剂活化，加入适量的茨菇抑制剂，在1～30℃条件下反应，在适合的缓冲溶液中放置12小时以上。茨菇抑制剂即可充分与载体材料交联。
本发明的亲和层析材料的应用：
茨菇抑制剂为两种多功能结晶蛋白酶抑制剂A和B的混合物，这两种蛋白酶抑制剂在抑制活性上略有不同，采用这种亲和层析材料在适合的PH条件下可用于除去蛋白质中所含的蛋白酶和激肽释放酶。
取约1000ml沉降体积的琼脂糖2B(Sephrose 2B，Pharmacia公司产品)用沙芯漏斗抽滤成半干物，用0.5mol/L氯化钠溶液洗涤，再用蒸馏水洗涤至中性。按每10ml沉降体积的琼脂糖2B与2g溴化氰的比例，称取溴化氰的研钵中碾磨，溶于0.1mol/L碳酸氢的溶液中。
将琼脂糖珠与溴化氰溶液的冰浴中混和搅拌，并滴加2mol/L氢氧化钠溶液，使pH保持在11.0～11.5之间，10分钟后移至室温放置10分钟。将混和液倒入布氏漏斗，并立即用0.1mol/L碳酸氢钠(pH9.5)缓冲溶液抽洗，在2～3分钟内洗下相当于琼脂糖珠10～15倍体积的缓冲溶液，终止活化。
按每1ml活化的凝胶可交联2.25mg的茨菇抑制剂计算，加入已调pH至9.5的茨菇抑制剂水溶液在4℃条件下缓慢搅拌2小时至少放置12小时。用约20倍体积的0.1mol/L碳酸氢钠(pH9.5)缓冲溶液洗涤已交联的琼脂糖珠，洗去未结合的蛋白。用0.1mol/L盐酸—甘氨酸缓冲溶液(pH2.4)洗去非特异性吸附蛋白。再用0.2mol/L甘氨酸缓冲溶液(pH8.5)封闭琼脂糖珠上与蛋白结合的活性基团，置4℃缓慢搅拌20小时再用0.1mol/L三羟甲基氨基甲烷-盐酸液(pH7.5)抽洗，即制得茨菇抑制剂—琼脂糖交联复合物。加20％乙醇置4℃保存备用。
实施实例II：
重复实施例I，用琼脂糖4B(Sephrose 4B，Pharmacia公司产品代替琼脂糖2B同样操作，制得茨菇抑制剂—琼脂糖4B交联复合物。
实施实例III：
重复实施例I，用琼脂糖6B(Sephrose 6B，Pharmacia公司产品)代替琼脂糖2B同样操作，制得茨菇抑制剂—琼脂糖6B交联复合物。
实施实例IV：
重复实施例I，用二乙基氨基乙基琼脂糖(DEAE-Sephrose，Pharmacia公司产品)代替琼脂糖2B同样操作，制得茨菇抑制剂一二乙基氨基乙基琼脂糖交联复合物。
实施实例V：
1.柱层析：
取采用以上实施实例方法制备的亲和层析材料各1000ml，装柱。事先用0.1mol/L(pH8.0)的磷酸缓冲溶液(含0.2mol/L的氯化钠)作为平衡液冲洗柱床至流出液电导与平衡液电导一致。另取尿激酶(比活力：50000IU/mg)制成浓度为100000IU/ml的溶液，调节pH至8.0。每次上样1000ml，上样完毕后，用1000ml的平衡液冲洗柱床，蛋白酶和激肽释放酶被亲和层析柱吸附，尿激酶则随溶液流出。层析柱可用0.1mol/L(pH4.0)醋酸钠缓冲溶液再生重复使用。
2.蛋白酶和激肽释放酶的测定：
(1)激肽释放酶的测定方法：采用发色底物S-2266测定法[入江章子等，临床药理(日本)29,419，(1981)]。
取0.2mol/L(pH8.0)三羟甲基氨基甲烷缓冲溶液500ul，37℃保温5～10分钟，再加入检品溶液400ul，混合37℃保温2～5分钟后加底物溶液100ul[25mgS-2266(日本第一化学制药株式会社产品)加28.8ml的蒸馏水溶解)混合37℃保温30分钟在加50％醋酸溶液终止反应。另取500ul的含抑肽酶10000～50000IU/L的0.2mol/L的三羟甲基氨基甲烷缓冲溶液(pH8.0)代替0.2mol/L的三羟甲基氨基甲烷缓冲溶液(pH8.0)同样操作，作为空白对照在滤长405nm处测定吸收度(A)，按下式计算激肽释放酶的含量：
激肽释放酶的含量(mU/ml)＝A×9.55
(2)蛋白酶的测定方法：酪蛋白分解活力测定法。
取1.0ml检品溶液加1.0ml的1％酪蛋白溶液混匀37℃保温1小时后，加入50％的三氯醋酸混匀沉淀未水解的蛋白质，3000rpm离心5分钟，弃去沉淀。另取1.0ml经100℃煮沸2～3分钟的检品溶液代替上述检品溶液同样操作，作为空白在280nm处测定吸收度(B)作为蛋白酶的活力(O.D.280/ml)。
(3)按上述方法测定尿激酶经亲和层析前后溶液中激肽释放酶和蛋白酶的含量(处理前：激肽释放酶2.1mU/ml，蛋白酶0.243O.D.280/ml)，测定结果见表一：
                           表一亲和层析柱               样品处理后                残存率(％)
             激肽释放酶    蛋白酶          激肽释放酶   蛋白酶
             (mU/ml)       (O.D.280/ml)M-Spherose 2B     0.0292        0.040          1.4          16.5M-Spherose 4B     0.0181        0.044          0.86         18.1M-Spherose 6B     0.0240        0.035          1.1          14.4M-DEAE-Spherose   0.0070        0.009          0.33         3.7M：代表茨菇抑制剂
实施实例VI：
重复实施实例V，用尿胰蛋白酶抑制剂(比活力：2500U/mg，含激肽释放酶10.5mU/ml，蛋白酶0.2300.D.280/ml)代替尿激酶制成浓度为50000U/ml的溶液，同样操作，测定结果见表二：
                         表二亲和层析柱               样品处理后             残存率(％)
               激肽释放酶  蛋白酶       激肽释放酶  蛋白酶
               (mU/ml)     (O.D.280/ml)M-Spherose 2B       0.0227      0.045        0.22       19.8M-Spherose 4B       0.0137      0.026        0.13       11.3M-Spherose 6B       0.0121      0.032        0.12       13.9M-DEAE-Spherose     0.0068      0.014        0.06       6.1M：代表茨菇抑制剂

Claims (3)

1、一种亲和层析材料，其特征在于其满足下列结构要求
R_茨菇抑制剂
R代表一种不溶于水的基质材料，包括各种类型的琼糖。
2、一种亲和层析材料的制备方法，其特征在于首先将不溶于水的基质材料R用溴化氰或其他激活剂活化，加入适量茨菇抑制剂，在1～30℃条件下反应，在适合的缓冲溶液中放置12小时以上，茨姑抑制剂即可充分与载体R交联。
3、一种亲和层析材料的应用，特征在于用权利要求1的亲和层析材料在适合的PH条件下去除蛋白质中所含的蛋白酶和激肽释放酶。
CN95114253A 1995-12-04 1995-12-04 一种亲和层析材料及制备方法和应用 Expired - Lifetime CN1057608C (zh)

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US7833723B2 (en) * 2006-01-06 2010-11-16 Millipore Corporation Affinity chromatography matrices and methods of making and using the same
WO2010111576A2 (en) * 2009-03-27 2010-09-30 Hercules Incorporated Aminated polymers and their use in water-borne compositions

Citations (2)

* Cited by examiner, † Cited by third party
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EP0203049A1 (en) * 1985-05-23 1986-11-26 Pharmacia Ab Method of cross-linking a porous polysaccharide gel
EP0295073A2 (en) * 1987-06-08 1988-12-14 Chromatochem, Inc. Chromatographic material

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0203049A1 (en) * 1985-05-23 1986-11-26 Pharmacia Ab Method of cross-linking a porous polysaccharide gel
EP0295073A2 (en) * 1987-06-08 1988-12-14 Chromatochem, Inc. Chromatographic material

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