朱桔Mn-SOD的纯化、鉴定及浓度梯度胶电泳在其中的应用

Purification and differentiation of Mn-SOD from Citrus erythrosa and application of gel concentration gradient electrophoresis

朱桔叶中存在Mn SOD、Fe SOD和CuZn SOD三种类型 ,在 1 0 %的PAGE中有三条活性带 ,其中Mn SOD的Rf值大于Fe SOD与CuZn SOD1 的Rf 值相同 ;在 4%～ 35 %的梯度胶电泳中有四条活性带 ,而Mn SOD的Rf 值最小 ,表明该酶带有较高的电荷密度。Mn SOD占总活性的2 0 %左右 ,已被纯化到均一程度。该酶的比活性为 1 2 4 9U/mg ,分子量和亚基分子量分别为5 4 .0kD和 2 6.6kD ,在紫外区最大吸收值为 2 80nm ,在 95℃处理 1 5min仍保留了 46%的酶活性 ,等电点为 5 .0 6。该酶的活性不被KCN、H2 O2 抑制 ,但对 1 %SDS和氯仿

There were Mn-SOD, Fe-SOD and CuZn-SOD in the crude extract of Citrus erythrosa Tanaka leaves. In a 10% PAGE three active bands were seen, and R f value of Mn-SOD was big than that of Fe-SOD and was same as that of CuZn-SOD 1. While in a 4%～35% gel concentration gradient electrophoresis, four active bands were seen and Mn-SOD's R f value was the smallest. This means that the molecular weight of Mn-SOD was the biggest and it had a high charge density. Mn-SOD, which occupied about 20% of the total SOD activity, was purified to homogeneity by means of ammonium sulfate precipitation, Sephadex G-100 gel filtration, DEAE-Sepharose and CM 52 chromatography etc. Its specific activity was 1 249 U/mg. Its molecular and subunit weight were 54.0 kD and 26.6 kD, respectively. It had one absorption maximum which was at 280 nm. And it was stable to heat treatment,46% of its activity was retained at 95℃ for 15 min. Isoelectric focusing electrophoresis showed that it had a pI of 5.06.This enzyme was insensitive to KCN and H 2O 2, but was sensitive to 1% SDS or mixtures of chloroform and ethanol(3:5 v/v).