pH值敏感相分离特性的蛋白质酶解试剂研究

pH Sensitive Phase Transition and Enzyme Activity of Trypsin-Chitosan Conjugant

通过DEC/NHS偶联反应将胰蛋白酶(Trypsin)连接到具有pH值敏感性的壳聚糖(CS)链上。Trypsin-CS偶联物保留了CS的pH值敏感相分离特性及Trypsin的酶催化活性。浊度滴定实验表明,此偶联物的临界相分离pH值(CPSP)仍保持在偏中性范围内,并且随CS的分子量降低而略有上升。这种偏中性的CPSP值有利于相分离循环过程中保持酶的催化活性。分子量为8×104的CS与Trypsin的偶联物在存放30d后可保留69%的初始酶活性。偶联物经过6次循环使用后的残余活性为初始活性的79%。Trypsin-CS偶联物可在低于CPSP值的条件下对蛋白质进行均相酶解,并在高于CPSP值时从催化体系中分离出来。HPLC肽谱分析表明,Trypsin-CS偶联物中Trypsin的自降解被显著抑制,因而有效消除了Trypsin自降解肽段对肽谱分析的干扰。

Trypsin-chitosan conjugant was prepared by coupling trypsin with chitosan（CS） through the method of 1-（3-dimethylaminopropyl）-3-ethylcarbodiimide/N-hudroxysuccinamide（EDC/NHS） coupling reactions.The resulting conjugant had borne both characteristics of pH-sensitive phase transitional behavior and enzyme catalytic activity.The critical phase transitional pH point（CPSP） of the conjugant was determined by turbid titration method.The results show that CPSP value increase with the decrease of chitosan molecular weight.The CPSP value of CS and its trypsin conjugant could reach 6.36 when the chitosan with molecular weight of 8 × 10^4 was used.The near neutral CPSP is in favor of stabilizing the enzyme activity during the cyclic procedure of phase transition.Trypsin-CS conjugant could preserve 69% of initial enzyme activity after 30 days storage,and 79% of enzyme activity was observed after 6 cyclic phase transitional procedure.Trypsin-CS conjugant could catalyze the degradation of BSA in homogeneous phase when the pH of solution is slightly less than CPSP value.The conjugant could be recovered by precipitating it from solution when the pH is higher than CPSP value.HPLC results demonstrated that the self degradation of trypsin was significantly inhibited and the interference from the self-degraded peptide could be eliminated in the peptide fragment mapping.