PubMed · 3131631
The source did not provide an abstract. Follow the original record for more information.
Explore connections, maps & timelines
Y Ikariyama, M Aizawa. 1988. Bioaffinity sensors.. https://doi.org/10.1016/0076-6879(88)37011-4
Cite the original work for its findings. Save a collection to share your selection of sources.
KEEP EXPLORING
We offer a new titration protocol for determining the dissociation constant and binding stoichiometry of protein-ligand complex, detectable by spectroscopic methods. This approach neither is limited to the range of protein or ligand concentrations employed during titration experiment nor relies on precise determinations of the titration "endpoint," i.e., the maximal signal changes upon saturation of protein by ligand (or vice versa). In this procedure, a fixed concentration of protein (or ligand) is titrated by increasing volumes of a stock ligand (or protein) solution, and the changes in the spectroscopic signal are recorded after each addition of the titrant. The signal for interaction between protein and ligand first increases, reaches a maximum value, and then starts decreasing due to dilution effect. The volume of the titrant required to achieve the maximum signal changes is utilized to calculate the dissociation constant and the binding stoichiometry of the protein-ligand complex according to the theoretical relationships developed herein. This procedure has been tested for the interaction of avidin with a chromophoric biotin analogue, 2-(4'-hydroxyazobenzene)benzoic acid by following the absorption signal of their interaction at 500 nm. The widespread applicability of this procedure to protein-ligand complexes detected by other spectroscopic techniques and its advantages over conventional methods are discussed.
Surface plasmon resonance detectors, such as the BIAcore instrument produced by Pharmacia, show promise for the detection and quantitation of macromolecular interactions in a label-free mode. Such detectors rely on the covalent immobilization of one of the interacting species onto the sensing surface. To date, the only published chemistry for this purpose is reaction of primary amino-containing ligands with an N-hydroxysuccinimide (NHS) ester-activated surface. In an effort to increase the versatility of the BIAcore with respect to immobilizing ligands, we undertook an investigation of activation chemistries compatible with this system. Using readily available reagents, we demonstrated that the carboxylated dextran-coated sensing surface could be easily converted to functions other than NHS-esters, including amine-activated, hydrazine-activated, and sulfhydryl-activated surfaces. In addition, use was made of the streptavidin/biotin interaction to probe chemical modifications of the sensing surface, by employing specifically modified biotin derivatives.
The streptavidin-biotin system has been used to immunotarget whole ricin to tumor cells in a system that overcomes ricin-nonspecific cytotoxicity. Biotin was linked to ricin via a disulfide-containing reagent, sulfosuccinimidyl-2-(biotinamido)ethyl-1,3'-dithiopropionate. The product, biotinyl-S,S-ricin (b-ricin), retained most of its in vitro cytotoxic activity against human epidermoid carcinoma (KB) cells. Complexing b-ricin to streptavidin resulted in greater than 99% loss of its cellular toxicity which is associated with loss of cell-binding activity. The streptavidin-b-ricin complex could, however, be targeted to KB cells via the biotinylated monoclonal antibody 108 which is specific to the epidermal growth factor receptor overexpressed on KB cells. The complex did not regain its activity if the specific antibody was not biotinylated or if the biotinylated antibody was of a different specificity. Streptavidin is thus used to block b-ricin, presumably due to a steric restraint of the streptavidin on the ricin B-chain, and to bridge it to biotinyl antibody recognizing the target cell. Avidin could not replace streptavidin in this system since a complex between b-ricin and avidin retained a major part (60%) of ricin cytotoxic activity. This is attributed to the nonspecific binding of avidin to cells in vitro, including the KB cells. It is suggested that b-ricin is blocked by both streptavidin and avidin, but once the complex gains access to the cell surface, its cytotoxic activity is specifically retrieved.