Freer Erik M, Yim Kang Sub, Fuller Gerald G, Radke Clayton J
Chemical Engineering Department, University of California, Berkeley, California 94720-1462, USA.
Langmuir. 2004 Nov 9;20(23):10159-67. doi: 10.1021/la0485226.
Proteins adsorbed at fluid/fluid interfaces influence many phenomena: food emulsion and foam stability (Murray et al. Langmuir 2002, 18, 9476 and Borbas et al. Colloids Surf., A 2003, 213, 93), two-phase enzyme catalysis (Cascao-Pereira et al. Biotechnol. Bioeng. 2003, 83, 498; 2002, 78, 595), human lung function (Lunkenheimer et al. Colloids Surf., A 1996, 114, 199; Wustneck et al.; and Banerjee et al. 2000, 15, 14), and cell membrane mechanical properties (Mohandas et al. 1994, 23, 787). Time scales important to these phenomena are broad, necessitating an understanding of the dynamics of biological macromolecules at interfaces. We utilize interfacial shear and dilatational deformations to study the rheology of a globular protein, lysozyme, and a disordered protein, beta-casein, at the hexadecane/water interface. Linear viscoelastic properties are measured using small amplitude oscillatory flow, stress relaxation after a sudden dilatational displacement, and shear creep response to probe the rheological response over broad experimental time scales. Our studies of lysozyme and beta-casein reveal that the interfacial dissipation mechanisms are strongly coupled to changes in the protein structure upon and after adsorption. For beta-casein, the interfacial response is fluidlike in shear deformation and is dominated by interfacial viscous dissipation, particularly at low frequencies. Conversely, the dilatational response of beta-casein is dominated by diffusion dissipation at low frequencies and viscous dissipation at higher frequencies (i.e., when the experimental time scale is faster than the characteristic time for diffusion). For lysozyme in shear deformation, the adsorbed protein layer is primarily elastic with only a weak frequency dependence. Similarly, the interfacial dilatational moduli change very little with frequency. In comparison to beta-casein, the frequency response of lysozyme does not change substantially after washing the protein from the bulk solution. Apparently, it is the irreversibly adsorbed fraction that dominates the dynamic rheological response for lysozyme. Using stress relaxation after a sudden dilatational displacement and shear creep response, the characteristic time of relaxation was found to be 1000 s in both modes of deformation. The very long relaxation time for lysozyme likely results from the formation of a glassy interfacial network. This network develops at high interfacial concentrations where the molecules are highly constrained because of conformation changes that prevent desorption.
吸附在流体/流体界面的蛋白质会影响许多现象：食品乳液和泡沫的稳定性（Murray等人，《朗缪尔》，2002年，18卷，9476页；Borbas等人，《胶体与界面科学》，A辑，2003年，213卷，93页）、两相酶催化（Cascao-Pereira等人，《生物技术与生物工程》，2003年，83卷，498页；2002年，78卷，595页）、人类肺功能（Lunkenheimer等人，《胶体与界面科学》，A辑，1996年，114卷，199页；Wustneck等人；以及Banerjee等人，2000年，15卷，14页）和细胞膜的力学性质（Mohandas等人，1994年，23卷，787页）。对这些现象重要的时间尺度范围很广，因此有必要了解生物大分子在界面处的动力学。我们利用界面剪切和拉伸变形来研究球状蛋白溶菌酶和无序蛋白β-酪蛋白在十六烷/水界面处的流变学。使用小振幅振荡流、突然拉伸位移后的应力松弛以及剪切蠕变响应来测量线性粘弹性性质，以探测在广泛实验时间尺度上的流变响应。我们对溶菌酶和β-酪蛋白的研究表明，界面耗散机制与吸附时和吸附后蛋白质结构的变化紧密相关。对于β-酪蛋白，其界面响应在剪切变形中类似流体，且以界面粘性耗散为主，尤其是在低频时。相反，β-酪蛋白的拉伸响应在低频时以扩散耗散为主，在高频时以粘性耗散为主（即当实验时间尺度快于扩散的特征时间时）。对于剪切变形中的溶菌酶，吸附的蛋白质层主要是弹性的，频率依赖性较弱。同样，界面拉伸模量随频率变化很小。与β-酪蛋白相比，从本体溶液中洗去蛋白质后，溶菌酶的频率响应变化不大。显然，对于溶菌酶，是不可逆吸附部分主导了动态流变响应。通过突然拉伸位移后的应力松弛和剪切蠕变响应，发现在两种变形模式下松弛的特征时间均为1000秒。溶菌酶极长的松弛时间可能是由于形成了玻璃态界面网络。这种网络在高界面浓度下形成，此时分子由于构象变化而高度受限，阻止了解吸。
