Teske Christopher A, Schroeder Magnus, Simon Robert, Hubbuch Jürgen
Institut für Biotechnologie 2, Forschungszentrum Jülich, Jülich 52425, Germany.
J Phys Chem B. 2005 Jul 21;109(28):13811-7. doi: 10.1021/jp050713+.
Confocal laser scanning microscopy (CLSM) is being increasingly used for observing protein uptake in porous chromatography resins. Recent CLSM studies have revealed the possible existence of a nondiffusive protein transport mechanism. Observing protein uptake with CLSM requires labeling the protein with a fluorescent probe. This study examines the effect of the probe identity on the subsequent CLSM adsorption profiles. The adsorption of lysozyme conjugated with different fluorescent probes (Cy5, BODIPY FL, Atto 635, and Atto 520) on SP Sepharose Fast Flow was measured using CLSM and zonal chromatography experiments. Results from zonal chromatography show that the retention time of lysozyme-dye conjugates differ significantly from unlabeled lysozyme. The change in retention of lysozyme upon conjugation with a fluorescent probe is consistent with the difference in net charge between the lysozyme-dye conjugate and unlabeled lysozyme. The adsorption profiles measured by CLSM show significantly different behavior depending upon whether the lysozyme-dye conjugate is retained longer or shorter than the unlabeled lysozyme. These results strongly suggest that the lysozyme concentration overshoot observed in previous CLSM experiments is the result of displacement of weaker binding labeled lysozyme by stronger binding unlabeled lysozyme.
共聚焦激光扫描显微镜（CLSM）越来越多地用于观察蛋白质在多孔色谱树脂中的摄取情况。最近的CLSM研究揭示了可能存在一种非扩散性蛋白质转运机制。用CLSM观察蛋白质摄取需要用荧光探针标记蛋白质。本研究考察了探针特性对后续CLSM吸附图谱的影响。使用CLSM和区带色谱实验测量了与不同荧光探针（Cy5、BODIPY FL、Atto 635和Atto 520）偶联的溶菌酶在SP Sepharose Fast Flow上的吸附情况。区带色谱结果表明，溶菌酶-染料偶联物的保留时间与未标记的溶菌酶有显著差异。溶菌酶与荧光探针偶联后保留情况的变化与溶菌酶-染料偶联物和未标记溶菌酶之间净电荷的差异一致。CLSM测量的吸附图谱显示出显著不同的行为，这取决于溶菌酶-染料偶联物的保留时间比未标记的溶菌酶长还是短。这些结果有力地表明，在先前的CLSM实验中观察到的溶菌酶浓度过冲是结合较弱的标记溶菌酶被结合较强的未标记溶菌酶取代的结果。
