Structural changes in hemoglobin during adsorption to solid surfaces:: Effects of pH, ionic strength, and ligand binding
Structural changes in hemoglobin during adsorption to solid surfaces:: Effects of pH, ionic strength, and ligand binding
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DOI：
10.1073/pnas.95.21.12271
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发表时间：
1998-10-13
影响因子：
11.1
通讯作者：
Brzezinski, P
中科院分区：
文献类型：
--
作者：
Höök, F;Rodahl, M;Brzezinski, P
我们用石英晶体微天平(QCM)技术研究了两种结构相似的血红蛋白(MET-Hb和HbCO)在金表面上的疏水自组装甲基末端硫醇单分子膜上的吸附。该技术允许以时间分辨的同时测量频率(F)(c.f.质量)和能量耗散(D)(参见QCM在吸附过程中的粘弹性)，这使得研究吸附过程中不同蛋白质层的粘弹性特性成为可能。在Met-Hb和HbCO等电点下方，Delta D-Delta f图显示了斜率显著不同的两个相，这表明吸附的蛋白质处于两种不同的粘弹性状态。第一相的斜率比第二相的斜率小，这表明第一相与较刚性结合的、可能是变性的蛋白质层结合有关，而第二相与形成第二层较松散结合的蛋白质有关。该第二层例如在吸附的MET-Hb的Fe3+还原并随后与一氧化碳(CO)结合形成HbCO时解吸。因此，结果表明，吸附在第二层的蛋白质处于类天然状态。这一信息只能通过同时对D和f的变化进行时间分辨测量来获得，这表明QCM技术提供了关于蛋白质在固体表面吸附机制的独特信息。
We have studied the adsorption of two structurally similar forms of hemoglobin (met-Hb and HbCO) to a hydrophobic self-assembled methyl-terminated thiol monolayer on a gold surface, by using a Quartz Crystal Microbalance (QCM) technique. This technique allows time-resolved simultaneous measurements of changes in frequency (f) (c.f. mass) and energy dissipation (D) (c.f. rigidity/viscoelastic properties) of the QCM during the adsorption process, which makes it possible to investigate the viscoelastic properties of the different protein layers during the adsorption process. Below the isoelectric points of both met-Hb and HbCO, the Delta D vs. Delta f graphs displayed two phases with significantly different slopes, which indicates two states of the adsorbed proteins with different visco-elastic properties. The slope of the first phase was smaller than that of the second phase, which indicates that the first phase was associated with binding of a more rigidly attached, presumably denatured protein layer, whereas the second phase was associated with formation of a second layer of more loosely bound proteins. This second layer desorbed, e.g., upon reduction of Fe3+ of adsorbed met-Hb and subsequent binding of carbon monoxide (CO) forming HbCO. Thus, the results suggest that the adsorbed proteins in the second layer were in a native-like state. This information could only be obtained from simultaneous, time-resolved measurements of changes in both D and f, demonstrating that the QCM technique provides unique information about the mechanisms of protein adsorption to solid surfaces.