超氧化物歧化酶固相修饰的研究

Study on Solid-Phase Modification of Superoxide Dismutase

对超氧化物歧化酶固相修饰进行初步研究。选择Q-Sepharose FF作为固相吸附介质,考察了吸附的影响因素。采用三聚氯氰活化好的单甲氧基聚乙二醇5 000分别对游离SOD和吸附在Q-Sepharose FF上的固相SOD进行化学修饰,考察了修饰的影响因素,并对两者进行了比较。Q-Sepharose FF吸附SOD的最佳条件:时间30 min,pH值9.0,SOD浓度3.0 mg/mL,最佳吸附条件下吸附量约为22.5 mg/mL;最佳固相修饰条件:时间270 min,修饰缓冲液浓度40 mmol/L,mPEG-5 000与SOD的摩尔比为50∶1,最佳固相修饰条件下固相SOD修饰率为38.3%,修饰后酶活为5 555 U/mg,酶活回收率为72.3%;经SDS-PAGE分析表明固相修饰可以减少修饰的随机性。

To conduct the preliminary research on solid-phase enzyme modification of superoxide dismutase, the Q-Sepharose FF was chosen as solid phase medium. Monomethoxypolyethylene glycol 5 000 was activated by cyanuric chloride, then combined covalently with free SOD and solid phase SOD respectively. The influential factors on modification was investigated and the optimum conditions of adsorption for free SOD （1） and solid SOD （2） were found as follows： the adsorption time was 30 min for （1） and 270 min for （2）, pH value was 9.0 for （1）, the concentration of SOD was 3.0 mg/mL for （1）, the maximum adsorption capacity could reach 22. 5 mg/mL for （1）. The concentration of borate buffer solution was 40 mmol/L for（2） and the molar ratio of mPEG-5 000 to SOD was 50 ： 1 for （2）. The experiments showed that the activity of prepared solid phase mPEG-SOD could keep about 72.3 % of the activity of the native SOD and its modification rate of amino residue was 38. 3 %. SDS-PAGE studies demonstrated that solid-phase enzyme modification could reduce random multi PEGylation.