Jackler Guido, Czeslik Claus, Steitz Roland, Royer Catherine A
Universität Dortmund, Physikalische Chemie I, D-44221 Dortmund, Germany.
Phys Rev E Stat Nonlin Soft Matter Phys. 2005 Apr;71(4 Pt 1):041912. doi: 10.1103/PhysRevE.71.041912. Epub 2005 Apr 27.
The spatial distribution of protein molecules interacting with a planar polyelectrolyte multilayer was determined using neutron reflectometry. Staphylococcal nuclease (SNase) was used as model protein that was adsorbed to the multilayer at 22 degrees C and 42 degrees C. At each temperature, the protein solution was adjusted to pD -values of 4.9 and 7.5 to vary the net charge of the protein molecules. The multilayer was built up on a silicon wafer by the deposition of poly(ethylene imine) (PEI), poly(styrene sulfonate) (PSS), and poly(allylamine hydrochloride) (PAH) in the order Si-PEI-PSS- (PAH-PSS)(5). Applying the contrast variation technique, two different neutron reflectivity curves were measured at each condition of temperature and pD -value. From the analysis of the curves, protein density profiles normal to the interface were recovered. Remarkably, it has been found that SNase is partially penetrating into the polyelectrolyte multilayer after adsorption at all conditions studied. The measured neutron reflectivities are consistent with a penetration depth of 50 A at pD=4.9 and 25 A at pD=7.5. Since SNase has an isoelectric point of pH=9.5, it carries a net positive charge at both pD -values and interacts with the PSS final layer under electrostatic attraction conditions. However, when increasing the temperature, the amount of adsorbed protein is increasing at both pD -values indicating the dominance of entropic driving forces for the protein adsorption. Interestingly, at pD=4.9 where the protein charge is relatively high, this temperature-induced mass increase of immobilized protein is more pronounced within the polyelectrolyte multilayer, whereas at pD=7.5, closer to the isoelectric point of SNase, raising the temperature has mainly the effect to accumulate protein molecules outside the polyelectrolyte multilayer at the water interface. It is suggested that the penetration of SNase into the polyelectrolyte multilayer is related to a complexation mechanism. The complexation is essentially entropic in nature due to the release of counterions.
利用中子反射技术测定了与平面聚电解质多层膜相互作用的蛋白质分子的空间分布。以葡萄球菌核酸酶（SNase）作为模型蛋白，在22℃和42℃下将其吸附到多层膜上。在每个温度下，将蛋白质溶液的pD值调节至4.9和7.5，以改变蛋白质分子的净电荷。通过在硅片上依次沉积聚（乙烯亚胺）（PEI）、聚（苯乙烯磺酸盐）（PSS）和聚（烯丙胺盐酸盐）（PAH），构建多层膜，顺序为Si-PEI-PSS-（PAH-PSS）（5）。应用对比变化技术，在每个温度和pD值条件下测量了两条不同的中子反射率曲线。通过对曲线的分析，得到了垂直于界面的蛋白质密度分布。值得注意的是，发现在所有研究条件下，吸附后SNase会部分渗透到聚电解质多层膜中。测得的中子反射率与pD = 4.9时50 Å和pD = 7.5时25 Å的渗透深度一致。由于SNase的等电点为pH = 9.5，它在两个pD值下都带净正电荷，并在静电吸引条件下与PSS终层相互作用。然而，当温度升高时，两个pD值下吸附的蛋白质量都在增加，这表明熵驱动力在蛋白质吸附中占主导地位。有趣的是，在pD = 4.9时蛋白质电荷相对较高，这种温度诱导的固定化蛋白质质量增加在聚电解质多层膜内更为明显，而在pD = 7.5时，更接近SNase的等电点，升高温度主要使蛋白质分子在水界面处聚集在聚电解质多层膜之外。有人认为，SNase渗透到聚电解质多层膜中与一种络合机制有关。由于抗衡离子的释放，这种络合本质上是熵驱动的。
