Model‐based development of an assay for the rapid detection of biotin‐blocked binding sites of streptavidin

The protein streptavidin (SAV) is applied in a large variety of molecular methods due to an extraordinarily strong binding to the vitamin biotin (BIO). The protein structure is homotetrameric, characterized by one binding site for BIO per subunit. Therefore, one of the major criteria to determine the quality of SAV isolates is the proportion of BIO-blocked binding sites per tetramer. A rapid analysis of BIO-free binding sites is achieved by fluorescence quenching of biotin-4-fluorescein (B4F). However, BIO-blocked binding sites can only be determined by costly and laborious procedures such as ELISA-based methods or radioactive labeling. This study describes the systematic, model-supported development of a method for the quick and simple detection of BIO-blocked binding sites, based on a short-term heat incubation of the sample in the presence of B4F. Kinetic modeling and parameter estimation yielded dissociation constants of 1.22 +/- 0.27 x 10(-11) M for the complex SAV-BIO and 5.16 +/- 0.70 x 10(-13) M for SAV-B4F at 70 degrees C, allowing a displacement of SAV-bound BIO by B4F. This method allows the rapid monitoring of BIO-blocked binding sites in fermentation processes, independent from the chain length of SAV and the concentration of contaminating proteins, e.g. when optimizing the BIO concentration in cultivation media.