摘要
将棕色固氮菌230含铁超氧化物歧化酶对8mol/L脲,10mmol/L EDTA透析制备无活性缺辅基蛋白;将其在8mol/L脲中对10mmol/L硫酸亚铁铵透析得到重组超氧化物歧化酶。重组酶含有与天然酶相近的铁含量,活性为天然酶的89.1％。缺辅基蛋白,重组酶与天然酶都是由二个相同的亚基组成;重组酶的吸收光谱与荧光光谱与天然酶几乎一样,而缺辅基蛋白则有较大的差异;从园二色谱的分析得知,缺辅基蛋白不含有α—螺旋,而天然酶和重组酶中α螺旋的含量分别为21％和20％;缺辅基蛋白比天然酶或重组酶具有更大的巯基反应性。
A stable metal-free apoenzyme has been obtained from Azotobacter Vin-elandii 230 iron-superoxide dismutase following exposure to 8 M urea in Tris-HCl buffer containing 10mM EDTA at acid pH and subsequently dialyse against neutral buffer to remove the urea. The reconstituted enzyme was obtained by incubation of the apoenzyme in 8 urea containing Tris-HCl buffer with the addition of ferrous ammonium sulfate.The apoenzyme had neither significant iron content nor enzymatic activity. The iron content of the reconstituted enzyme was nearly the same as that of the native one, but only 89.1% of enzymatic activity was recovered. The apoenzyme and the reconstituted enzyme were dimeric in structure as the native one. The UV absorption and fluorescence spectra of the apoenzyme were different from that of the native enzyme. But the reconstituted enzyme had similar UV absorption and fluorescence spectra as the native one. The sulfhydryl groups of the apoenzyme showed high reactivity with 5,5'-dithiobis-(2-nitrobenzoic acid), while those of the native and reconstituted enzyme had little reactivity. Circular dichroism spectra of the reconstituted enzyme in 200-300 nm region were similar in shape to that of the native one, but obviously different from that of the apoenzyme. The a-helical contents of the native and reconstituted enzymes were estimated to be 21% and20%,respectively. However, the apoenzyme seemed to be lack of a-heliex. Thermal in-activation kinetics studies showed that the reconstituted enzyme was less stable than the native enzyme.
出处
《生物化学杂志》
CAS
CSCD
1989年第5期411-416,共6页
关键词
棕色固氮菌
FE-SOD
重组酶
Iron-Superoxide dismutase Azotobacter vinelandii Reconstituted enzyme Apoenzyme
