重组人锰超氧化物歧化酶的胃蛋白酶酶解特性及其稳定性

Pepsin digestion characteristics and stability of recombinant human manganese-superoxide dismutase

目的探讨重组人锰超氧化物歧化酶(rhMn-SOD)的胃蛋白酶酶解特性及其稳定性。方法在37℃条件下,用模拟胃液(0.084 mol/L HCl,35 mmol/L NaCl,pH 2.0,4 000 U胃蛋白酶)处理rhMn-SOD不同时间,SDS-PAGE分析酶解效果;使用总SOD测定试剂盒检测rhMn-SOD在不同温度(25、37、45、55、65、75℃)下保存不同时间(10、20、30、40、50、60 min)、不同pH(4.0～10.0)、不同化合物(氯仿、甘油、DMSO、10%SDS、β-巯基乙醇、苯酚、30%H2O2)处理后及日光和紫外照射后的相对活力。结果 rhMn-SOD在模拟胃液中处理2 min即可完全酶解。在25～55℃范围内,rhMn-SOD具有较好的热稳定性,在65～75℃范围内,rhMn-SOD的热稳定性较差;在pH 6.0～8.0范围内,rhMn-SOD具有较好的稳定性;rhMn-SOD对甘油和氯仿不敏感,经DMSO、SDS处理后,活力大幅下降,经β-巯基乙醇、苯酚、30%H2O2处理后,基本失活;rhMn-SOD经日光和紫外照射3 h,活力基本不变。结论分析了rhMnSOD的胃蛋白酶酶解特性、热稳定性、pH稳定性、对不同化合物的敏感性以及光稳定性,为其广泛应用于化妆品、药品、食品添加剂等领域奠定了基础。

Objective To investigate the pepsin digestion characteristics and stability of recombinant human manganese- superoxide dismutase （rhMn-SOD）. Methods The rhMn-SOD was treated with mock gastric juice （0. 084 mol / L hy- drochloric acid, 35 mmol/L sodium chloride, pH 2. 0, 4 000 U pepsin） at 37 ℃ for various minutes, analyzed for diges- tion effect by SDS-PAGE. The relative activities of rhMn-SOD after storage at various temperatures（25, 37, 45, 55, 67 and 75 ℃） for various minutes （10, 20, 30, 40, 50 and 60 min） and at various pH values （4. 0 - 10. 0）, after treatment with various compounds （chloramines, glycerol, DMSO, 10% SDS, β-mercaptoethanol, phenol, 30% hydrogen peroxide） and after exposure to sunlight and ultraviolet irradiation were determined by total SOD determination kit. Results The rhMn- SOD was completely digested after treatment with mock gastric juice for 2 min, which showed high thermostability at 25 ~ 55 ~C and low thermostability at 65 - 75 ~C. The rhMn-SOD showed high stability at pH 6. 0 - 8. 0, and was insensitive to glycerol and chloramines, of which the activity decreased remarkably after treatment with DMSO and SDS and disap- peared basically after treatment with β-mercaptoethanol, phenol and 30% hydrogen peroxide. However, the activity of rhMn-SOD showed no significant change after exposure to sunlight or to ultraviolet irradiation for 3 h. Conclusion The pepsin digestion characteristics, thermostability, stability to pH value, sensitivities to various compounds as well as photo- stability of rhMn-SOD were analyzed, which laid a foundation of its application in various fields such as cosmetics, drugs and food additives.