Parvanova Katja, Idakieva Krassimira, Todinova Svetla, Genov Nicolay
Institute of Organic Chemistry, Bulgarian Academy of Sciences, Akad. G. Bonchev-Str. Bl. 9, 1113 Sofia, Bulgaria.
Biochim Biophys Acta. 2003 Sep 23;1651(1-2):153-62. doi: 10.1016/s1570-9639(03)00265-6.
Rapana thomasiana hemocyanin (RtH) is a mixture of two hemocyanin (Hc) isoforms termed RtH1 and RtH2. Both subunit types are built up of eight functional units (FUs). The C-terminal functional unit (RtH1-h) of the Rapana Hc subunit 1 has been isolated by limited trypsinolysis of the subunit polypeptide chain. The oxy- and apo-forms of the unit are characterized by fluorescence spectroscopy. Upon excitation of RtH1-h at 295 or 280 nm, tryptophyl residues buried in the hydrophobic interior of the protein globule determine the fluorescence emission. This is confirmed by quenching experiments with acrylamide, cesium chloride and potassium iodide. The copper-dioxygen system at the binuclear active site quenches the indole emission of the oxy-RtH1-h. The removal of this system increases the fluorescence quantum yield and causes structural rearrangement of the microenvironment of the emitting tryptophyl residues in the apo-RtH1-h. The thermal stability of the apo-RtH1-h is characterized fluorimetrically by the "melting" temperature T(m) (65 degrees C) and by the transition temperature T(m) (83 degrees C) obtained by differential scanning calorimetry for oxy-RtH1-h. The results confirm the role of the copper-dioxygen complex for the stabilization of the Hc structure in solution.
香螺血蓝蛋白（RtH）是由两种血蓝蛋白（Hc）亚型组成的混合物，分别称为RtH1和RtH2。两种亚基类型均由八个功能单元（FU）组成。香螺血蓝蛋白亚基1的C末端功能单元（RtH1-h）通过对亚基多肽链进行有限的胰蛋白酶消化而分离出来。该功能单元的氧合形式和脱辅基形式通过荧光光谱进行表征。当在295或280nm激发RtH1-h时，埋在蛋白质球疏水内部的色氨酸残基决定了荧光发射。这通过丙烯酰胺、氯化铯和碘化钾的猝灭实验得到证实。双核活性位点的铜-双氧体系猝灭了氧合RtH1-h的吲哚发射。去除该体系会增加荧光量子产率，并导致脱辅基RtH1-h中发射色氨酸残基微环境的结构重排。脱辅基RtH1-h的热稳定性通过荧光法以“熔解”温度T(m)（65℃）以及通过差示扫描量热法得到的氧合RtH1-h的转变温度T(m)（83℃）来表征。结果证实了铜-双氧配合物在溶液中对血蓝蛋白结构稳定的作用。
