Biosensors and Bioelectronics, October 2012, Vol.38(1), pp.74-78
We have developed a technique for sensing protein–small molecule and protein–ion interactions in bulk aqueous solution by utilizing a pH sensitive dye, 5-(and-6)-carboxyfluorescein, conjugated to free lysine residues on the surfaces of designated capture proteins. The fluorescein intensity was found to change by about 6% and 15% for small molecule and ion binding, respectively. The assay works by modulating the local electric fields around a pH sensitive dye. This, in turn, alters the dye's apparent pK value. Such changes may result directly from the charge on the analyte, occur through allosteric effects related to the binding process, or result from a combination of both. The assay was used to follow the binding of Ca to calmodulin (CaM) and thiamine monophosphate (ThMP) to thiamine binding protein A (TbpA). The results demonstrate a binding constant of 1.1 μM for the Ca /CaM pair and 3.2 nM for ThMP/TbpA pair, which are in excellent agreement with literature values. These assays demonstrate the generality of this method for observing the interactions of small molecules and ions with capture proteins. In fact, the assay should work as a biosensor platform for most proteins containing a specific ligand binding site, which would be useful as a simple and rapid preliminary screen of protein–ligand interactions. ► pH modulation sensing detects the binding of small molecules and ions to proteins. ► The optical biosensor developed is a rapid method for screening ligand–receptor binding. ► The method developed is general and can be used with most proteins containing a binding site.
Biosensor ; Fluorescein ; Protein ; Small Molecule ; Ph Sensitive ; Labeling ; Engineering ; Biology
View record in ScienceDirect (Access to full text may be restricted)