血管紧张素转换酶固相化方法的选择  

Immobilization methods of angiotensin converting enzyme

作　　者：刘宏[1] 陈兰英[1] 

机构地区：[1]中国协和医科大学中国医学科学院心血管病研究所阜外心血管病医院,北京100037

出　　处：《基础医学与临床》2001年第3期278-281,共4页Basic and Clinical Medicine

基　　金：国家新药研究基金 (96 - 90 1- 0 5 - 85 )

摘　　要：分别采用琼脂包埋法、卵清包埋法、海藻酸钙包埋法、明胶 -戊二醛包埋法、溴化氰活化连接法和苯甲磺酰氯结合方法对血管紧张素转换酶进行固相化研究 ,发现苯甲磺酰氯结合法用于血管紧张素转换酶固相化效果最好。此实验是在低水活度条件下 ,利用苯甲磺酰氯在丙酮和吡啶存在的条件下活化SepharoseCL 4B凝胶侧链基团上的羟基 ,形成具有高反应活性的苯甲磺酰基团 ,在pH7 8的 0 2mol/LHEPES HCl缓冲系统中 ,4℃反应 12h ,将酶与凝胶连接在一起。 2 5mgACE与 5g活化琼脂糖凝胶在这种条件下反应 ,所得固相酶活力为 10 86U/mg ,蛋白固相率达到 6 6 % ,酶活力固相率为 5 3 3%。固相酶 4℃保存 3个月 ,活力剩余 80 % ,2 0℃保存 1个月酶活残留 6 1%。同样条件下 ,溶液酶活力分别只剩余 6 2 %和 19% ,由此可见血管紧张素转换酶经固相化后稳定性明显提高。The immobilization of angiotensin converting enzyme (ACE) was carried out by agarose investment, ovi albumin investment, alginic acid calcium investment, bromine cyan activation and tosylate chloride activation methods, the results of detecting immobilized ACE activity indicated that among these methods, the tosylate chloride method was the most effective one The reaction was performed under waterless condition, using tosylate chloride to active side chain hydroxyl group of Sepharose CL 4B agarose, formed a high activity group which could react with free amino group of ACE at 0 2mol/L HEPES HCl buffer, pH7 8, gently shaking 12h at 4℃, thus linked enzyme with agarose 25mg ACE and 5g activated agarose linked under such condition, immobilized ACE activity was 10 86U/g dry agarose, the enzyme protein immobilization rate was 66% and the enzyme activity immobilization rate reached 53 3% After stored in 4℃ for 3 months or in 20℃ for a month, the activity of immobilized enzyme remained 80% and 61% respectively, in contrast with this the activity of soluble enzyme had remained 62% and 19% respectively So it could conclude that immobilized enzyme had a better stability than solvable enzyme, this would be helpful for further investigate

关 键 词：血管紧张素转换酶 SEPHAROSE CL-4B 苯甲磺酰氯 固相化 

分 类 号：R341[医药卫生—基础医学]