Agustinho S C, Tinto M H, Imasato H, Tominaga T T, Perussi J R, Tabak M
Departamento Química e Física Molecular, Instituto de Química de São Carlos, Universidade de São Paulo, Brazil.
Biochim Biophys Acta. 1996 Dec 5;1298(2):148-58. doi: 10.1016/s0167-4838(96)00127-6.
Sephadex G-200 chromatography of the extracellular hemoglobin from the giant earthworm G. paulistus in the met form presents a single peak at pH 7.0 and two peaks at pH 9.0 as a result of alkaline dissociation. SDS-PAGE shows that the polypeptide chains are very similar to those observed for the oxy form and the two peaks at pH 9.0 correspond to the trimer contaminated by linkers and monomers which seems to be quite pure. The aquomet acid form is stable as an oligomer of molecular mass 3.1 x 10(6) Da only in a narrow pH range around neutrality. Increasing the pH above 7.5 leads to an irreversible transition from aquomet to hemichrome I which is the low-spin bis-imidazole complex. At pHs above 9.5-10.0 a second reversible transition takes place from hemichrome I to hemichrome II, a high-spin complex which is associated with the weakening and possible disruption of the proximal Fe--N histidine bond. Thus, increase in pH above 8.0 induces changes in the heme pocket that involve both the distal and proximal sides of the heme. EPR measurements show a very sharp decrease of the aquomet high-spin signal in the range of pH 7.0-8.0 and a very small low-spin signal even at liquid helium temperatures. The transition to hemichrome I is also accompanied by the loss of heme optical activity monitored by CD, which is consistent with the weakening of heme--globin interaction. Hemichrome I in the presence of cyanide gives the typical cyanometHb derivative which has a transition to a hemichrome at much higher pHs. This observation suggests that the dissociation of the oligomer in alkaline medium as well as the stability of the heme on the proximal side, depend both upon the ligand present at the sixth coordination position on the distal side. Hence, we believe that hemi(hemo)chrome formation in G. paulistus Hb and other invertebrate hemoglobins is a common phenomenon, not associated with protein denaturation, which may provide a fine tuning mechanism to control subunit interactions through changes in the distal side of the heme pocket.
来自巨型蚯蚓圣保罗巨蚓（G. paulistus）的变构形式的细胞外血红蛋白在pH 7.0时经葡聚糖凝胶G - 200层析呈现单峰，而在pH 9.0时由于碱性解离呈现双峰。SDS - PAGE显示，多肽链与氧合形式所观察到的非常相似，并且在pH 9.0时的两个峰对应于被连接体污染的三聚体和似乎相当纯的单体。高铁酸形式仅在接近中性的狭窄pH范围内作为分子量为3.1×10(6) Da的寡聚体稳定。将pH提高到7.5以上会导致从高铁酸不可逆地转变为高铁血红素I，即低自旋双咪唑配合物。在pH高于9.5 - 10.0时，会发生从高铁血红素I到高铁血红素II的第二次可逆转变，高铁血红素II是一种高自旋配合物，与近端铁 - 氮组氨酸键的减弱和可能的断裂有关。因此，pH高于8.0时的升高会引起血红素口袋的变化，这涉及血红素的远端和近端两侧。电子顺磁共振测量显示，在pH 7.0 - 8.0范围内高铁酸高自旋信号急剧下降，甚至在液氦温度下也有非常小的低自旋信号。向高铁血红素I的转变还伴随着通过圆二色性监测的血红素光学活性的丧失，这与血红素 - 珠蛋白相互作用的减弱一致。在氰化物存在下的高铁血红素I产生典型的氰化高铁血红蛋白衍生物，该衍生物在高得多的pH值下转变为高铁血红素。这一观察结果表明，碱性介质中寡聚体的解离以及血红素在近端一侧的稳定性，都取决于远端第六配位位置上存在的配体。因此，我们认为圣保罗巨蚓血红蛋白和其他无脊椎动物血红蛋白中高铁（血红）素的形成是一种常见现象，与蛋白质变性无关，这可能提供一种微调机制，通过血红素口袋远端的变化来控制亚基相互作用。
