Zemła Joanna, Lekka Małgorzata, Wiltowska-Zuber Joanna, Budkowski Andrzej, Rysz Jakub, Raczkowska Joanna
Smoluchowski Institute of Physics, Jagellonian University, Reymonta 4, Kraków, Poland.
Langmuir. 2008 Sep 16;24(18):10253-8. doi: 10.1021/la801313u. Epub 2008 Aug 16.
Most methods developed to study protein binding to distinct surfaces can only determine the average amount of adsorbed protein or merely provide (qualitative) information on its spatial distribution. Both these features can be characterized rigorously by integral geometry analysis of fluorescence micrographs. This approach is introduced here to compare the relative protein adsorption onto various polymer surfaces: polystyrene (PS), poly(methyl methacrylate) (PMMA), poly( n-butyl methacrylate) (PnBMA), poly( tert-butyl methacrylate) (PtBMA), and PS(PETA) and cross-linked poly(ethylene oxide) (PEO*(PETA)), admixed with pentaerythritol triacrylate (PETA). The polymeric surfaces were incubated for 15 min in phosphate-buffered saline (pH 7.4) containing 125 mug/mL fluorescently labeled lectins, either lentil lectin (LcH) or concanavalin A (ConA). Fluorescence images were recorded at identical conditions (physiological buffer, same exposure time, magnification, gain). For each image, taken a few times for each polymer, the distribution and average value of the normalized intensity were determined. The results show that the binding of LcH to PS(PETA), PtBMA, PS, PnBMA, PMMA, and PEO*(PETA) can be expressed by the ratio of the following values (mean +/- 95% confidence interval): 0.356 +/- 0.022, 0.298 +/- 0.030, 0.241 +/- 0.014, 0.083 +/- 0.008, 0.039 +/- 0.008, and 0.010 +/- 0.006, respectively. In turn, the relative adsorption of ConA is described by the values 0.252 +/- 0.016, 0.217 +/- 0.014, 0.222 +/- 0.016, 0.046 +/- 0.006, 0.116 +/- 0.008, and 0.006 +/- 0.002, respectively. Low dispersions of fluorescence intensity around average values indicate homogeneous distribution of adsorbed proteins. The introduced approach enables a fast and easy way not only to quantify the relative amount of bound proteins but also to characterize quantitatively the organization of their surface distribution, as demonstrated for patchlike protein adsorption onto the polymer blend surface.
大多数用于研究蛋白质与不同表面结合的方法，只能确定吸附蛋白质的平均量，或者仅仅提供关于其空间分布的（定性）信息。通过对荧光显微照片进行积分几何分析，可以严格地表征这两个特征。本文引入这种方法来比较蛋白质在各种聚合物表面上的相对吸附情况：聚苯乙烯（PS）、聚甲基丙烯酸甲酯（PMMA）、聚甲基丙烯酸正丁酯（PnBMA）、聚甲基丙烯酸叔丁酯（PtBMA）、PS(PETA)以及与季戊四醇三丙烯酸酯（PETA）混合的交联聚环氧乙烷（PEO*(PETA)）。将这些聚合物表面在含有125μg/mL荧光标记凝集素（扁豆凝集素（LcH）或伴刀豆球蛋白A（ConA））的磷酸盐缓冲盐水（pH 7.4）中孵育15分钟。在相同条件下（生理缓冲液、相同曝光时间、放大倍数、增益）记录荧光图像。对于每种聚合物拍摄几次的每张图像，确定归一化强度的分布和平均值。结果表明，LcH与PS(PETA)、PtBMA、PS、PnBMA、PMMA和PEO*(PETA)的结合可以用以下值的比率表示（平均值±95%置信区间）：分别为0.356±0.022、0.298±0.030、0.241±0.014、0.083±0.008、0.039±0.008和0.010±0.006。相应地，ConA的相对吸附分别由以下值描述：0.252±0.016、0.217±0.014、0.222±0.016、0.046±0.006、0.116±0.008和0.006±0.002。荧光强度在平均值周围的低离散度表明吸附蛋白质的分布均匀。如聚合物共混物表面上斑块状蛋白质吸附的示例所示，引入的方法不仅提供了一种快速简便的方式来量化结合蛋白质的相对量，还能定量地表征其表面分布的组织情况。
