Topham R W, Tesh S, Bonaventura C, Bonaventura J
Marine Biomedical and Biotechnology Center, Duke University Marine Laboratory, Beaufort, North Carolina 28516.
Arch Biochem Biophys. 1988 Mar;261(2):299-311. doi: 10.1016/0003-9861(88)90345-1.
Previously reported differences in the reactivities toward active-site ligands such as hydrogen peroxide indicate that the active-site geometries of the arthropod and mollusc hemocyanins are significantly different. Results are presented which demonstrate that the purified subunits composing the native hemocyanin of an arthropod show comparable active-site heterogeneity. Neither whole nor stripped samples of Limulus oxyhemocyanin are completely oxidized by reaction with hydrogen peroxide. Moreover, the addition of hydrogen peroxide to deoxy or completely oxidized samples causes partial regeneration to the oxy form. This behavior is most pronounced for one of the five major chromatographic fractions, Zone III, which retains greater than 60% of its copper-oxygen absorbance band when treated with hydrogen peroxide. Zone III is composed of three subunits (IIIA, IIIB, and IIIB'). With excess hydrogen peroxide, the deoxy and oxidized forms of Subunit IIIA can be completely regenerated to a fully functional oxy state, while Subunits IIIB and IIIB' are less than 30% regenerated. Upon long-term storage, Subunit IIIA slowly loses 340 nm absorbance, an aging process that can be reversed by hydrogen peroxide. These features of Subunit IIIA, that set it apart from other subunits of the 48-member ensemble of intact Limulus hemocyanin, are typically exhibited by mollusc hemocyanins rather than arthropod hemocyanins. These findings clearly illustrate that significantly different active-site geometries can occur within a single type of metalloprotein and suggest that comparisons between the various Limulus subunits and Subunit IIIA may prove valuable in unraveling the structural basis for the disparity in the chemical reactivity exhibited by hemocyanins of different species.
先前报道的对诸如过氧化氢等活性位点配体的反应性差异表明，节肢动物和软体动物血蓝蛋白的活性位点几何结构存在显著差异。本文给出的结果表明，构成节肢动物天然血蓝蛋白的纯化亚基表现出相当的活性位点异质性。鲎氧合血蓝蛋白的完整样品或脱辅基样品与过氧化氢反应时都不会完全被氧化。此外，向脱氧或完全氧化的样品中添加过氧化氢会使其部分再生为氧合形式。这种行为在五个主要色谱馏分之一的区域III中最为明显，该馏分在用过氧化氢处理时保留了其铜 - 氧吸收带的60%以上。区域III由三个亚基（IIIA、IIIB和IIIB'）组成。在过量过氧化氢存在下，亚基IIIA的脱氧形式和氧化形式可以完全再生为功能完全正常的氧合状态，而亚基IIIB和IIIB'的再生率不到30%。长期储存后，亚基IIIA会缓慢失去340 nm的吸光度，这一老化过程可被过氧化氢逆转。亚基IIIA的这些特性使其有别于完整鲎血蓝蛋白48个亚基组成的集合中的其他亚基，这些特性通常是软体动物血蓝蛋白而非节肢动物血蓝蛋白所具有的。这些发现清楚地表明，在单一类型的金属蛋白中可能存在显著不同的活性位点几何结构，并表明比较各种鲎亚基和亚基IIIA可能有助于揭示不同物种血蓝蛋白所表现出的化学反应性差异的结构基础。
