不同 pH 和温度对 A 型肉毒毒素结构及活性的作用

摘要

Objective To study the effects of pH and temperature on structure and activity of botulinum toxin type A. Methods The botulinum toxin type A was dissolved in pH 6.6 and 7.8 PB then stored at 4℃and 35℃for 30 days, re-spectively.HPLC and mouse bioassay were used to investigate the changes on structure and activity of botulinum toxin type A.Results The HPLC sample 1 (pH6.6,4 ℃) diagram showed only peak I (complex) and it’s activity did not reduce noticeablly on the 30th day;the HPLC sample 2 ( pH7.8,4 ℃) diagram showed peak II ( neurotoxin) dissociated from peak I as the sample was initially dissolved in pH 7.8 buffer, and peak II proportion was gradually increased along with in-creasing of time, but this had no effect on it’ s activity on the 30th day; the HPLC sample 3 ( pH6.6 35 ℃) diagram showed only peak I initially, then peak II appeared on the 7th day, and peak III appeared on the 21st day associated with a sharply reduced activity (50%of original activity), the activity reduced gradually to 40% of original activity on the 30th day;The HPLC sample 4(pH7.8,35 ℃) diagram showed peak I, peak II, and peak III on the 1st day associated with a sharply reduced activity (50%of original activity), the activity reduced to 50%of original activity on the 30th day.Con-clusion Acid condition is helpful to maintain the configuration of complex of botulinum toxin type A, alkaline condition could destroy the non-covalent bond between the complex components.Whether in acidic or alkaline condition, the high temperature will accelerate the dissociation of complex and breaking down disulfide bond on components of neurotoxin, by which the toxin activity will be reduced.%目的：研究pH和温度对A型肉毒毒素结构及活性的影响。方法将A型肉毒毒素溶解于pH6．6和pH7．8的磷酸盐缓冲液（PB）中，分别采用4℃和35℃两种温度保存30天，在不同时间采用高压液相色谱（HPLC）及小鼠试验研究其结构及生物学活性的变化。结果样品1（pH6．6，4℃）的HPLC图谱30天内只呈现Ⅰ峰（复合物），毒力未见明显下降；样品2（pH7．8，4℃）则在溶解的最初便有Ⅱ峰（神经毒素）解离出来，并且随着时间的增加Ⅱ峰所占比例逐渐增大，毒力在30天内未见明显下降；样品3（pH6．6，35℃）最初只呈现Ⅰ峰，在第7天便有Ⅱ峰解离出来，第21天开始Ⅲ峰（轻链）出现并伴随着毒力的大幅下降（最初毒力的50％），30天降低至最初毒力的约40％；样品4（pH7．8，35℃）在第1天便有Ⅰ、Ⅱ、Ⅲ峰出现，伴随着毒力的大幅下降（最初毒力的50％），30天降低至最初毒力的约25％。结论采用酸性条件有利于维持A型肉毒毒素复合体的结构，碱性会破坏复合体各组分间的非共价键。不论用酸性还是碱性条件，高温度会加速复合体的解离与神经毒素二硫键的断裂，二硫键的断裂导致活性的下降。