化学发光法测定超氧化物歧化酶

Detection of superoxide dismutase activities by chemilumiscence.

采用海萤萤光素诱导剂（ＣＬＡ，２－Ｍｅｔｈｙｌ－６－ｐｈｅｎｙｌ－３，７－ｄｉｈｙｄｒｏｉｍｉｄａｚｏ－［ｌ，２－α］ｐｙｒａｚｉｎｅ－３－ｏｎｅ）作为化学发光剂，以黄嘌呤－黄嘌呤氧化酶体系产生超氧阴离子，用ＫＣＮ络合铜锌离子，使铜锌超氧化物歧化酶（Ｃｕ、Ｚｎ－ＳＯＤ）失活，建立了检测血浆及红细胞ＳＯＤ酶活性的ＣＬＡ化学发光法。本法检测活力为５０００Ｕ／ｍｇ标准ＳＯＤ的ＩＣ（５０）＝０．０３２９μｇ／ｍｌ，正常混合人血浆ＩＣ５０＝０．７１μｌ，标准测定的平均回收率为１０３．２％，测试标准液批内ＣＶ为２．６％，混合人血浆的批内ＣＶ为３．５％，用此法测得的健康成人血浆ＳＯＤ总活力为２６３±４９×ｌ０３Ｕ／Ｌ，锰超氧化物歧化酶（Ｍｎ－ＳＯＤ）活力为８２±１６×１０３Ｕ／Ｌ，Ｃｕ、Ｚｎ－ＳＯＤ活力为１５８±３４×１０３Ｕ／Ｌ，红细胞ＳＯＤ活力为５７８４±２００１Ｕ／ｇＨｂ（ｘ±ｓ）。

Superoxide dismutase(SOD)activities derived from human plasma and erythrocytes can be quantifiedby the inhibitory effect of SOD.Using cypridina luciferin attractant(CLA,2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-α]-pyrazine-3-one),the chemiluminescence induced by a xanthine-xanthine oxidase system(a superoxide radi-cal ion generating system)was measured.Mn-SOD activity was measured with KCN to inactivate Cu· ZnSOD activity. The method is very sensitive(IC(50)=0.0329μg/ml,SOD=5000units/mg of protein. IC(50)=0. 71μl,human plasma.).It is accurate(rate of recovery 103.2%,on the average)and has good reproducibility(standardSOD,CV2.6%,human plasma, CV3.5%).It is simple and rapid.Using the proposed methed,T-SOD activityis 263±49×103U/L,Mn-SOD 82±16×103U/L,Cu·Zn-SOD 158±34×103U/L in the plasma of normal humanand SOD activity is 5784±2001U/g Hb(x±s)in the erythrocytes.