Harrington J P, Hirsch R E
Department of Chemistry, University of South Alabama, Mobile 36688.
Biochim Biophys Acta. 1991 Feb 15;1076(3):351-8. doi: 10.1016/0167-4838(91)90475-f.
The steady-state fluorescence properties of the multisubunit hemoglobin isolated from the earthworm, Lumbricus terrestris, were studied by front-face fluorometry. Acid and alkaline dissociation of this high-molecular-weight hemoglobin were examined over the pH range 3.7-12.5 using different liganded states (oxy, CO, met). The relative intensity of the emission maximum at 320 nm (exc. 280 nm) is ligand-dependent increasing as follows: oxy less than deoxy less than CO less than met at pH 7.0. The intensity of the emission maximum of oxyhemoglobin at the alkaline acid end point, pH 10.5 (333 nm), is significantly greater than that observed at the acid end point, pH 4.18 (320 nm), suggesting different subunit dissociation. The spectra of oxyhemoglobin at pH 4.18 and the spectrum of carbonmonoxy hemoglobin at pH 7.0 in the presence of 1 M magnesium chloride were almost identical, indicating similar subunit dissociation. Difference spectrum (pH 9.0-7.2) of fluorescence emission (exc. 305) resulted in a maximum at 341 nm, indicative of tyrosinate formation. This suggests that tyrosine(s) may also be located at the subunit interface(s) of this hemoglobin. These studies indicate that several aromatic amino acid residues are associated with the critical sites of subunit interactions within this molecule. Analysis of the fluorescence spectra also suggests that the formation of different subunit species resulting from acid and alkaline dissociation cannot be ruled out.
采用前表面荧光法研究了从蚯蚓（地龙）中分离出的多亚基血红蛋白的稳态荧光特性。使用不同的配体状态（氧合、一氧化碳结合、高铁），在pH值3.7 - 12.5范围内检测了这种高分子量血红蛋白的酸碱解离情况。在pH 7.0时，320 nm（激发波长280 nm）处发射峰的相对强度与配体有关，其增加顺序如下：氧合态＜脱氧态＜一氧化碳结合态＜高铁态。氧合血红蛋白在碱性终点pH 10.5（333 nm）处发射峰的强度明显大于在酸性终点pH 4.18（320 nm）处观察到的强度，表明亚基解离情况不同。在1 M氯化镁存在下，pH 4.18时氧合血红蛋白的光谱与pH 7.0时碳氧血红蛋白的光谱几乎相同，表明亚基解离情况相似。荧光发射（激发波长305）的差示光谱（pH 9.0 - 7.2）在341 nm处出现最大值，表明酪氨酸盐形成。这表明酪氨酸可能也位于这种血红蛋白的亚基界面处。这些研究表明，几个芳香族氨基酸残基与该分子内亚基相互作用的关键位点相关。对荧光光谱的分析还表明，不能排除酸碱解离导致形成不同亚基种类的可能性。
