title: Electrophoretic heterogeneity limits the utility of streptavidin-β-galactosidase as a probe in free zone capillary electrophoresis separations.
abstract: 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) cm(2)V(-1)s(-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.
authors: Chemistry Department, University of Winnipeg, 599 Portage Avenue, Winnipeg, MB, Canada. d.craig@uwinnipeg.ca
authors: Douglas B
authors: DB
authors: Craig
authors: Anna
authors: A
authors: Henderson
mesh_terms: D055162:Biocatalysis; D004269:DNA, Bacterial / Q000737:chemistry; D019075:Electrophoresis, Capillary / Q000379:methods*; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D019809:Streptavidin / Q000737:chemistry*; D001616:beta-Galactosidase / Q000737:chemistry*
chemical_list: D004269:DNA, Bacterial; D019809:Streptavidin; D001616:beta-Galactosidase