Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., Bl. 9, Sofia 1113, Bulgaria.
Comp Biochem Physiol B Biochem Mol Biol. 2013 Mar;164(3):201-9. doi: 10.1016/j.cbpb.2012.12.007. Epub 2013 Jan 11.
The intrinsic and inducible o-diphenoloxidase (o-diPO) activity of Cancer pagurus hemocyanin (CpH) and Limulus polyphemus hemocyanin (LpH) were studied using catechol, l-Dopa and dopamine as substrates. The kinetic analysis shows that dopamine is a more specific substrate for CpH than catechol and l-Dopa (K(m) value of 0.01 mM for dopamine versus 0.67 mM for catechol, and 2.14 mM for l-Dopa), while k(cat) is highest for catechol (2.44 min(-1) versus 0.67 min(-1) for l-Dopa and 0.71 min(-1) for dopamine). On treatment with 4mM sodium dodecyl sulfate (SDS) or by proteolysis the o-diPO activity of CpH increases about twofold. In contrast, native LpH shows no o-diPO activity, and exhibits only a slight activity after incubation with SDS. Neither CpH nor LpH show intrinsic mono-PO activity with l-tyrosine and tyramine as substrates. To explore the possible correlation between the degree of PO activity and protein stability of arthropod hemocyanins, the thermal stability of CpH and LpH was investigated by differential scanning calorimetry and Fourier transform infrared spectroscopy. CpH is found to be less thermostable (T(m)80 °C), suggesting that the dicopper active sites are more accessible, thereby allowing the hemocyanin to show PO activity in the native state. The LpH, on the other hand, is more thermostable (T(m)92 °C), suggesting the existence of a correlation between the thermal stability and the intrinsic PO activity of arthropod hemocyanins.
使用儿茶酚、L-多巴和多巴胺作为底物，研究了十足目甲壳动物血蓝蛋白（CpH）和鲎血蓝蛋白（LpH）的固有和诱导的邻二酚氧化酶（o-DiPO）活性。动力学分析表明，多巴胺是 CpH 比儿茶酚和 L-多巴更特异的底物（多巴胺的 K(m)值为 0.01mM，儿茶酚为 0.67mM，L-多巴为 2.14mM），而 k(cat)值最高的是儿茶酚（2.44min(-1)比 L-多巴的 0.67min(-1)和多巴胺的 0.71min(-1)）。用 4mM 十二烷基硫酸钠（SDS）处理或蛋白水解后，CpH 的 o-DiPO 活性增加约两倍。相比之下，天然 LpH 没有 o-DiPO 活性，用 SDS 孵育后仅表现出轻微的活性。CpH 和 LpH 均不以 L-酪氨酸和酪胺为底物表现出固有的单 PO 活性。为了探讨 PO 活性程度与节肢动物血蓝蛋白蛋白质稳定性之间的可能相关性，通过差示扫描量热法和傅里叶变换红外光谱法研究了 CpH 和 LpH 的热稳定性。发现 CpH 的热稳定性较低（T(m)80°C），这表明双铜活性位点更容易接近，从而使血蓝蛋白在天然状态下表现出 PO 活性。另一方面，LpH 的热稳定性较高（T(m)92°C），这表明节肢动物血蓝蛋白的热稳定性与固有 PO 活性之间存在相关性。
