Yang C C, Chang L S
Institute of Life Sciences, National Tsing Hua University, Hsinchu, Taiwan, Republic of China.
Biochem J. 1991 Dec 15;280 ( Pt 3)(Pt 3):739-44. doi: 10.1042/bj2800739.
Notexin from Notechis scutatus scutatus snake venom was subjected to tyrosine modification with p-nitrobenzenesulphonyl fluoride (NBSF), and four modified derivatives were separated by h.p.l.c. The results of amino acid analysis and sequence determination revealed that only Tyr-7, Tyr-70 and Tyr-77 were modified in notexin. Modification of Tyr-7 resulted in decreases in lethal toxicity and enzymic activity by 70.2% and 22.7% respectively. Conversely, modification of Tyr-77 caused a 1.8-fold increase in enzymic activity, in contrast with the loss of 52.5% of lethality. A drastic decrease in lethal toxicity was observed when both Tyr-7 and Tyr-70 were modified, whereas the enzymic activity decreased by only 35.8%. Likewise, the derivative in which Tyr-7 and Tyr-77 were modified retained 44.4% of enzymic activity, but showed a marked decrease in lethal toxicity. It is obvious that modification of tyrosine residues causes a decrease in lethal toxicity of notexin, which does not directly correlate with the change in enzymic activity. On the other hand, the antigenicity of NBS derivatives remained unchanged. The modified derivatives retained their affinity for Ca2+, indicating that the modified tyrosine residues did not participate in Ca2+ binding. These results indicate that modification of tyrosine residues can differentially influence the enzymic activity and lethal toxicity of notexin, and suggest that notexin might possess two functional sites, one being responsible for the catalytic activity and the other associated with its lethal effect.
对盾鳞棘背蛇（Notechis scutatus scutatus）蛇毒中的诺维毒素（notexin）用对硝基苯磺酰氟（NBSF）进行酪氨酸修饰，并通过高效液相色谱法（h.p.l.c.）分离出四种修饰衍生物。氨基酸分析和序列测定结果表明，诺维毒素中只有Tyr-7、Tyr-70和Tyr-77被修饰。Tyr-7的修饰导致致死毒性和酶活性分别降低70.2%和22.7%。相反，Tyr-77的修饰使酶活性增加了1.8倍，而致死率损失了52.5%。当Tyr-7和Tyr-70都被修饰时，观察到致死毒性急剧下降，而酶活性仅下降了35.8%。同样，Tyr-7和Tyr-77被修饰的衍生物保留了44.4%的酶活性，但致死毒性明显降低。显然，酪氨酸残基的修饰会导致诺维毒素致死毒性降低，这与酶活性的变化没有直接关联。另一方面，NBS衍生物的抗原性保持不变。修饰后的衍生物保留了对Ca2+的亲和力，表明修饰后的酪氨酸残基不参与Ca2+结合。这些结果表明，酪氨酸残基的修饰可以不同程度地影响诺维毒素的酶活性和致死毒性，并表明诺维毒素可能具有两个功能位点，一个负责催化活性，另一个与其致死效应相关。
