朱桔Mn-SOD的纯化、鉴定及浓度梯度胶电泳在其中的应用

朱桔叶中存在Mn-SOD、Fe-SOD和CuZn-SOD三种类型,在10%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R_f值最小,表明该酶带有较高的电荷密度。Mn-SOD占总活性的20%左右,已被纯化到均一程度。该酶的比活性为1 249 U/mg,分子量和亚基分子量分别为54.0 kD和26.6 kD,在紫外区最大吸收值为280 nm,在95℃处理15 min仍保留了46%的酶活性,等电点为5.06。该酶的活性不被KCN、H₂O₂抑制,但对1% SDS和氯仿-乙醇液敏感。

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

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 fvalue of Mn-SOD was big than that of Fe-SOD and was same as that of CuZn-SOD₁.While in a 4%~35% gel concentration gradient electrophoresis,four active bands were seen and Mn-SOD's R fvalue 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 CMsub>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 H₂O₂,but was sensitive to 1% SDS or mixtures of chloroform and ethanol (3:5 v/v).