聚丙烯酰胺梯度凝胶电泳和均一凝胶电泳在分离及鉴定植物SOD同工酶中的比较研究

Comparison of Gradient Polyacrylamide and Polyacrylamide Gel Electrophoresis in the Isolation and Identification of Plant SOD Isozyme

比较了聚丙烯酰胺梯度凝胶电泳(GPGE)和均一凝胶电泳(PAGE)在分离和鉴定SOD同工酶方面的应用.在GPGE中根据Rf值大小可将SOD谱带分为3个区:A区Rf值最小,对SDS敏感,为Mn-SOD;C区Rf值最大,对SDS不敏感,为CuZn-SOD;B区谱带Rf值位于两者之间,对SDS敏感,为Fe-SOD.根据GPGE图谱上谱带Rf值大小和对SDS敏感性来判断SOD类型结果与用抑制剂判断结果一致,它可作为一种SOD类型初步鉴定的简便可行的方法,相同植物材料经PAGE分离后不具备这些特性.在相同牛血SOD浓度下,GPGE检测SOD灵敏度可达10-15mol,PAGE为10-13mol;在1.56×10-12molSOD时,在GPGE中可分辨出4条带,PAGE仅为2条.枸杞Fe-SOD在7.5%胶中Rf值最大,在10%胶中与CuZn-SOD重叠,香橼Fe-SOD、Mn-SOD和朱桔Mn-SOD、CuZn-SOD在10%PAGE中相互重叠,在GPGE中它们都有规律地完全分离,可根据Rf值大小鉴别SOD类型.通过2次电泳阐明了GPGE分离和鉴定SOD的可靠性,它能有效地分离和鉴定在PAGE上重叠的3种SOD类型.

This paper compared the application of gradient polyacrylamide gel electrophresis （GPGE） and polyacrylamide gel eleetrophoresis（PAGE） in isolation and identification of plant SOD isozyme. The results showed that SOD bands on GPGE were divided into three region of A, B, C according to its Rf. The Rf of A region SOD band was minimum, it was sensitive to 1% SDS and was Mn-SOD. The Rf of C region SOD band was the biggest, it was not sensitive to 1% SDS and was CuZn-SOD. The Rf of B region SOD band was between A and C, it was sensitive to 1% SDS and was Fe-SOD. The three SOD isozymes were distinguished by its Rf and sensitivity to SDS on polyacrylamide gradient gel, this result was about the same as the result of judgement using inhibitor. The same plant sample was isolated by PAGE did not have the characteristic. 10^-13mol and 10^-15mol bovine SOD was detected in PAGE and GPGE, respectively. Four and two SOD activity bands were detected in GPGE and PAGE at 1.56 × 10^- 12 mol bovine SOD, respectively. The Rf of Fe-SOD from Lycium chinense was the biggest in 7. 5% PAGE and it overlapped with CuZn-SOD in 10% PAGE. The FeSOD and Mn-SOD of cirus wilsonii, Mn-SOD and CuZn-SOD of Citrus erythrosa were overlapped each other in 10% PAGE, but three types of SOD were completely isolated in 4% - 35% GPGE. The reliability of isolating and identify SOD using GPGE was expounded by two times gel concentration gradient electrophoresis TGCGE, the Rf and the number of SOD bands in GPGE were compatible with TGCGE, and it can completely isolate and identify the overlapped three SOD types on PAGE.