Wu Xiaoyu, Narsimhan Ganesan
Biochemical and Food Process Engineering, Department of Agricultural and Biological Engineering, Purdue University, West Lafayette, Indiana 47907, USA.
Langmuir. 2008 May 6;24(9):4989-98. doi: 10.1021/la703349c. Epub 2008 Mar 27.
Nanoparticles possess unique properties as a result of their large surface area per unit volume and therefore can be functionalized by the immobilization of enzymes for a variety of biosensing applications. Changes in the tertiary conformation of beta-lactoglobulin adsorbed on 90 nm silica nanoparticles with time were inferred using tryptophan fluorescence and Fourier transform infrared spectroscopy (FTIR) for different surface concentrations, temperature, pH, ionic strength, and 2,2,2-trifluoroethanol (TFE) and dithiothreitol (DTT) concentrations. Rapid initial unfolding followed by a much slower rate at longer times was observed, with the extent of unfolding being higher at lower surface concentrations, higher ionic strengths, higher temperature, higher TFE and DTT concentrations, and pI. The effect of temperature on the unfolding of adsorbed protein on the nanoparticle surface was similar to that in the bulk even though the extent of unfolding was higher for adsorbed protein molecules. The results of the extent of change in tertiary conformation using FTIR as indicated by the change in the ratio of amide II'/amide I were consistent with those obtained by tryptophan fluorescence whereas the rates of conformational changes given by FTIR were found to be much faster. Circular dichroism (CD) spectra showed that altering the surface concentration by itself did not change the secondary structure of beta-lactoglobulin on the surface. TFE was found to increase the alpha helix content at the expense of the fraction of the beta sheet, whereas the beta sheet was converted to an unordered conformation in the presence of DTT.
由于纳米颗粒每单位体积具有较大的表面积，因此具有独特的性质，从而可以通过固定酶来进行功能化，以用于各种生物传感应用。利用色氨酸荧光和傅里叶变换红外光谱（FTIR），推断了不同表面浓度、温度、pH、离子强度以及2,2,2-三氟乙醇（TFE）和二硫苏糖醇（DTT）浓度下，吸附在90 nm二氧化硅纳米颗粒上的β-乳球蛋白三级构象随时间的变化。观察到初始时快速展开，随后在较长时间内速率慢得多，在较低表面浓度、较高离子强度、较高温度、较高TFE和DTT浓度以及pI条件下，展开程度更高。温度对纳米颗粒表面吸附蛋白展开的影响与本体中的情况相似，尽管吸附蛋白分子的展开程度更高。使用FTIR得到的三级构象变化程度的结果，如酰胺II'/酰胺I比值的变化所示，与色氨酸荧光得到的结果一致，而FTIR给出的构象变化速率要快得多。圆二色性（CD）光谱表明，仅改变表面浓度并不会改变表面β-乳球蛋白的二级结构。发现TFE会增加α-螺旋含量，同时β-折叠部分减少，而在DTT存在的情况下，β-折叠会转变为无序构象。
