Schütz J, Dolashka-Angelova P, Abrashev R, Nicolov P, Voelter W
abteilung für Physikalische Biochemie des Physiologisch-chemischen Instituts der Universität Tübingen, Hoppe-Seyler-Strasse 4, D-72076 Tübingen, Germany.
Biochim Biophys Acta. 2001 Apr 7;1546(2):325-36. doi: 10.1016/s0167-4838(01)00152-2.
Keyhole limpet hemocyanin is a respiratory glycoprotein of high molecular weight from the gastropod mollusc Megathura crenulata. Two subunits, KLH1 and KLH2, were isolated using ion exchange chromatography and their physical properties are compared with the parent molecule. The various proteins are characterized by fluorescence spectroscopy, combined with fluorescence quenching studies, using acrylamide, cesium chloride and potassium iodide as tryptophan quenchers. The conformational stability of the native aggregate and its isolated structural subunits are also studied by circular dichroism and fluorescence spectroscopy as a function of temperature, as well as in the presence of guanidinium hydrochloride and urea. The associated subunits in the hemocyanin aggregates increase considerably the melting temperature to 67 degrees C and the free energy of stabilization in water, DeltaG(H(2)O)(D), towards guanidinium hydrochloride is higher for the decamer as compared to the isolated subunits; this difference can be accounted for by the stabilizing effects of intra-subunit interactions exerted within the oligomer. The copper-dioxygen complex at the active site additionally stabilizes the molecule, and removing of the copper ions increases the tryptophan emission and the quantum yield of the fluorescence.
钥孔血蓝蛋白是一种来自腹足纲软体动物巨缘帽贝的高分子量呼吸糖蛋白。使用离子交换色谱法分离出两个亚基KLH1和KLH2，并将它们的物理性质与母体分子进行比较。使用丙烯酰胺、氯化铯和碘化钾作为色氨酸猝灭剂，通过荧光光谱结合荧光猝灭研究对各种蛋白质进行表征。还通过圆二色性和荧光光谱研究了天然聚集体及其分离的结构亚基的构象稳定性，作为温度的函数，以及在盐酸胍和尿素存在的情况下。血蓝蛋白聚集体中的相关亚基显著提高了熔解温度至67℃，并且与分离的亚基相比，十聚体在水中的稳定自由能ΔG(H₂O)(D)对盐酸胍更高；这种差异可以通过寡聚体内亚基间相互作用的稳定作用来解释。活性位点的铜-双氧络合物进一步稳定了分子，去除铜离子会增加色氨酸发射和荧光量子产率。
