Linden T, Ljunglöf A, Kula M R, Thömmes J
Institut für Enzymtechnologie, Heinrich-Heine Universität Düsseldorf, 52426 Jülich, Germany.
Biotechnol Bioeng. 1999 Dec 20;65(6):622-30.
The use of confocal scanning laser microscopy (CSLM) has recently been described for the visualization of intraparticle protein profiles during single-protein finite bath uptake experiments. By coupling of fluorescent molecules to proteins the penetration of porous media by labeled macromolecules could be detected by scanning single adsorbent particles for fluorescence emission after laser excitation. Thus the internal protein distribution profile, which is a central element in modeling of protein transport in porous adsorbents, became experimentally accessible. Results from the simultaneous visualization of two proteins by this technology are shown here. The use of two different fluorescent dyes for protein labeling and two independent detectors in the CSLM allowed for the first time ever the direct observation of a two-component diffusion process within a porous stationary phase. The finite bath uptake of human immunoglobulin G (hIgG) and bovine serum albumin (BSA) to two different ion exchange adsorbents (SP Sepharose Fast Flow and Source 30S) and to an affinity adsorbent (Protein A Sepharose) was measured using Cy5 and Oregon Green as labels. Single adsorbent particles were scanned for intensity distribution of fluorescence emission from the two fluorophors. The intraparticle profiles obtained from the confocal images were translated into a relative protein concentration thus allowing the calculation of protein uptake kinetics from direct measurement in the stationary phase. The confocal technique may prove to be a very powerful means of data generation for modeling of multi-component mass transfer phenomena in protein adsorption.
最近，共聚焦扫描激光显微镜（CSLM）已被用于在单蛋白有限浴吸收实验中可视化颗粒内蛋白质分布。通过将荧光分子与蛋白质偶联，在激光激发后扫描单个吸附剂颗粒的荧光发射，可检测标记大分子在多孔介质中的渗透情况。因此，内部蛋白质分布情况（这是多孔吸附剂中蛋白质传输模型的核心要素）在实验中变得可获取。本文展示了利用该技术同时可视化两种蛋白质的结果。在CSLM中使用两种不同的荧光染料对蛋白质进行标记以及两个独立的探测器，首次实现了对多孔固定相内双组分扩散过程的直接观测。使用Cy5和俄勒冈绿作为标记，测量了人免疫球蛋白G（hIgG）和牛血清白蛋白（BSA）对两种不同离子交换吸附剂（SP Sepharose Fast Flow和Source 30S）以及一种亲和吸附剂（Protein A Sepharose）的有限浴吸收情况。扫描单个吸附剂颗粒以获取两种荧光团的荧光发射强度分布。从共聚焦图像获得的颗粒内分布情况被转化为相对蛋白质浓度，从而能够根据固定相中的直接测量结果计算蛋白质吸收动力学。共聚焦技术可能被证明是一种非常强大的数据生成手段，用于蛋白质吸附中多组分传质现象的建模。
