Electrophoretic Heterogeneity Limits the Utility of Streptavidin-β-Galactosidase as a Probe in Free Zone Capillary Electrophoresis Separations
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Single molecule assays were performed on streptavidin-β-galactosidase using a capillary electrophoresis-based protocol in order to assess the suitability of single molecule β-galactosidase assays for adaptation to the detection of single copies of target DNA. The conjugate was found to have a heterogeneous catalytic rate, showing an average rate of 44,000 ± 24,000 min−1, which is similar to that of the unmodified enzyme. Electrophoretic mobility was also measured on individual molecules and determined to be −1.32 × 10−4 ± 0.19 × 10−4 cm2V−1s−1. The variance in mobility was several times that reported for the unmodified enzyme. The electrophoretic heterogeneity was found to result in the formation of a broad window of peaks in the resultant electropherograms of free zone separations of small plugs of streptavidin-β-galactosidase. This range of mobilities largely overlapped with that of the conjugate bound to primer and plasmid containing a target DNA sequence. This overlap suggests that the separation of free conjugate from that bound to target DNA, which is a requirement for application of the single enzyme molecule assay to the detection of target DNA sequences, is not plausible using free zone capillary electrophoresis.
KeywordsAvidin conjugate Biotinylated primer Capillary electrophoresis Catalytic heterogeneity Electrophoretic mobility Electrophoretic heterogeneity Fluorescence detection β-galactosidase Separation Single molecule Streptavidin conjugate Target DNA
This study was supported by a grant from the Natural Sciences and Engineering Research Council.
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