抗冻蛋白作为高效冷冻保护剂对易冷变性的LDH保护效果观察

Observation of the protective effect of antifreeze protein as protectant on freeze denaturation of lactate dehydrogenase

目的探讨抗冻蛋白(AFP)对易冷变性的乳酸脱氢酶(LDH)保护效果。方法在LDH样本加入AFP、BSA、PVP设置成分别含0.001 mg/L、0.01 mg/L、0.1 mg/L、1 mg/L AFP,0.1 mg/L、1.0 mg/L、10 mg/L BSA以及0.1 mg/L、1.0 mg/L、10 mg/L PVP为添加剂的试验样本,依次记为AFP1、AFP2、AFP3、AFP4、BSA1、BSA2、BSA3、PVP1、PVP2、PVP3组。样本中加入等量0.02 mol/L p H 7.4 Tris-HCl缓冲液作为对照。各组设5个平行管。采用程序降温仪进行冻融试验,从25℃开始以20℃/min降温速度降温至-60℃,然后进行解冻融化升温至25℃。观察5个冻融循环后各试样LDH活性改变。结果与冻融前比较,对照组LDH活性恢复率为69.6%±4.1%,BSA1、PVP1、AFP1组LDH活性恢复率分别为75.2%±4.5%、73.3%±3.8%、71.8%±3.5%,差异有统计学意义(均P<0.01)。其他各组在冷融后酶活性恢复较好,仅出现轻度下降,差异无统计学意义(P>0.05)。添加0.01 mg/L AFP、1.0 mg/L BSA、1.0 mg/L PVP即有保护作用。结论 AFP对防止LDH冷变性有更好的保护效果。

Objective To research the protective effect of antifreeze protein（AFP） on freeze denaturation of lactate dehydrogen- ase （LDH）. Methods AFP, BSA and PVP were added respectively into the LDH samples to configurate different test＇sam- pies, and the concentrations of the additive were 0. 001 mg/L, 0.01 mg/L, 0.1 mg/L, 1 mg/L AFP, 0.1 mg/L, 1.0 mg/L, 10 mg/L BSA and 0.1 mg/L, 1.0 mg/L, 10 mg/L PVP respectively, marked as AFP1, AFF2, AFP3, AFP4, BSA1, BSA2, BSA3, PVP1, PVP2, PVP3 respectively. Buffer solutions of O. 02 moi./L pH 7.4 Tris - HCl was added to sample as control group. Five parallel pipes were set up in each group. A pre - programmed freezing - thawing cycle was applied to the samples in computer - controlled programmable freeze - thawing test, starting from 25℃ and cooling down to - 60℃ at the rate of 20 ℃/min, then back to 25℃ to thaw, thus the circle was repeated. The LDH activity change of samples before and after 5 freeze - thawing cycles Was observed. Results After 5 freeze - thawing cycles, compared with pre - freeze - thawing sam- pies, the activity recovery of LDH of control group was 69.6% ±4.1% , the activity recoveries of LDH of BSA1 group, PVP1 group, and AFP1 group were 75.2% ±4.5%, 73.3% ± 3.8%, 71.8% ± 3.5% respectively （P 〈0.01 ）, the differences were statistically significant. The activity recoveries of the LDH of the other groups were better, only slight declined, but the differences weren＇t significantly, P 〉0.05. Additive of 0.01 mg,/L AFP, 1.0 mg/L BSA, 1.0 mg,/L PVP could protect freeze denaturation of LDH. Conclusion Antifrezee protein was better cryoprotectant to protect LDH from freeze denaturation.