O'Malley S M, McDonald M J
Department of Chemistry, College of Arts and Sciences, University of Massachusetts, Lowell 01854.
J Protein Chem. 1994 Oct;13(7):585-90. doi: 10.1007/BF01890456.
The acrylamide quenching of the intrinsic tryptophanyl fluorescence of normal and sickle beta apohemoglobins has been studied in 0.05 M potassium phosphate buffer, pH 7.5, at 5 degrees C over a protein concentration range from 1 to 50 microM. Analysis of quenching dynamics revealed a strong dependence on acrylamide concentration for the intrinsic fluorescence of both normal and sickle beta apohemoglobins, suggesting that one tryptophanyl residue [presumably that at position 37(C3)], was more accessible to collisional quencher than the other beta tryptophanyl residue [15(A12)]. Additional studies, which altered viscosity and subunit assembly experimental parameters, supported the assignment of residue 37 as the more dynamically accessible residue. Finally, the quenching data were also found to be dependent on protein concentration, implying that this difference in the mobility between the two residues is a sensitive probe of self-aggregation. Extrapolated dynamic quenching constants at low concentration of acrylamide were used to estimate the dimer-monomer equilibrium dissociation constants of normal and sickle beta apohemoglobins, and were found to be 5.6 and 2.4 microM, respectively, thus demonstrating distinct self-association properties of beta A and beta S apohemoglobins.
在0.05 M磷酸钾缓冲液（pH 7.5）中，于5℃下，研究了丙烯酰胺对正常和镰刀型β-脱辅基血红蛋白内在色氨酸荧光的猝灭作用，蛋白质浓度范围为1至50 μM。猝灭动力学分析表明，正常和镰刀型β-脱辅基血红蛋白的内在荧光对丙烯酰胺浓度有很强的依赖性，这表明一个色氨酸残基[可能是37位（C3）的那个]比另一个β-色氨酸残基[15位（A12）]更容易被碰撞猝灭剂接近。改变粘度和亚基组装实验参数的进一步研究支持将37位残基确定为更具动态可及性的残基。最后，还发现猝灭数据依赖于蛋白质浓度，这意味着两个残基之间这种迁移率的差异是自聚集的敏感探针。在低丙烯酰胺浓度下外推得到的动态猝灭常数用于估计正常和镰刀型β-脱辅基血红蛋白的二聚体-单体平衡解离常数，结果分别为5.6和2.4 μM，从而证明了βA和βS脱辅基血红蛋白具有不同的自缔合特性。
