Dolashka-Angelova P, Hristova R, Schuetz J, Stoeva S, Schwarz H, Voelter W
Institute of Organic Chemistry, Bulgarian Academy of Sciences, Sofia.
Spectrochim Acta A Mol Biomol Spectrosc. 2000 Sep;56A(10):1985-99. doi: 10.1016/s1386-1425(99)00273-5.
The dodecameric hemocyanin of the crab Maia squinado contains five major electrophoretically separable polypeptide chains (structural subunits) which have been purified by FPLC ion exchange chromatography. The various proteins have been characterized by fluorescence spectroscopy, combined with fluorescence quenching studies, using acrylamide, caesium chloride and potassium iodide as tryptophan quenchers. The results show that the tryptophyl side chains of dodecameric Hc are deeply buried in hydrophobic regions of the hemocyanin aggregates and the quenching efficiency values for the native Hc in comparison with those from the constituent subunits are two to four times less. The conformational stabilities of the native dodecameric aggregate and its isolated structural subunits towards various denaturants (pH, temperature, guanidinium hydrochloride) indicate that the quaternary structure is stabilized by hydrophilic and polar forces, whereby, both, the oxy- and apo-forms of the protein have been considered. The critical temperatures for the structural subunits, Tc, determined by fluorescence spectroscopy, are in the region of 50-60 degrees C, coinciding with the melting temperatures, Tm, determined by CD spectroscopy. The free energy of stabilization in water, deltaG(D)H2O, toward guanidinium hydrochloride is about two times higher for the dodecamer as compared to the isolated subunits. These studies reveal that oligomerization between functional subunits has a stabilizing effect on the whole molecule and differences in the primary structures result in different stabilities of the subunits.
黄道蟹的十二聚体血蓝蛋白包含五条主要的可通过电泳分离的多肽链（结构亚基），这些亚基已通过快速蛋白质液相色谱离子交换色谱法纯化。利用丙烯酰胺、氯化铯和碘化钾作为色氨酸淬灭剂，通过荧光光谱结合荧光淬灭研究对各种蛋白质进行了表征。结果表明，十二聚体血蓝蛋白的色氨酸侧链深埋于血蓝蛋白聚集体的疏水区域，与组成亚基相比，天然血蓝蛋白的淬灭效率值低两到四倍。天然十二聚体聚集体及其分离的结构亚基对各种变性剂（pH、温度、盐酸胍）的构象稳定性表明，四级结构由亲水性和极性作用力稳定，其中考虑了蛋白质的氧合形式和脱辅基形式。通过荧光光谱测定的结构亚基的临界温度Tc在50 - 60摄氏度范围内，与通过圆二色光谱测定的解链温度Tm相符。与分离的亚基相比，十二聚体对盐酸胍在水中的稳定化自由能ΔG(D)H2O大约高两倍。这些研究表明，功能亚基之间的寡聚化对整个分子具有稳定作用，一级结构的差异导致亚基具有不同的稳定性。
