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 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.
Similar content being viewed by others
Abbreviations
- CE:
-
Capillary electrophoresis
- DDAO:
-
7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one)
- DDAO-gal:
-
9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) β-D-galactopyranoside
- LIF:
-
Laser-induced fluorescence
- PMT:
-
Photomultiplier tube
- strep-β-gal:
-
Streptavidin-β-galactosidase conjugate
References
Alam J, Cook JL (1990) Anal Biochem 188:245–254
Cano RJ, Torres MJ, Klem RE, Palomares JC, Casadesus J (1992) J Appl Bacteriol 72:393–399
Craig DB, Arriaga EA, Wong JCY, Lu H, Dovichi NJ (1996) J Am Chem Soc 118:5245–5253
Chen D, Dovichi NJ (1996) Anal Chem 68:690–696
Craig D, Dovichi NJ (1998) Can J Chem 76:623–626
Craig DB, Morris TT, Ong-Justiniano CMQ (2012) Anal Chem 84:4598–4602
Craig DB, Nachtigal JT, Ash HL, Shoemaker GK, Dyck AC, Wawrykow TMJ, Gudbjartson HL (2003) J Protein Chem 22:555–561
Craig DB, Schwab T, Sterner R (2012) Biochem Cell Biol 90:540–547
Dhar TK, Dasgupta S, Ray D, Banerjee M (2012) J Immunol Methods 385:71–78
Edman L, Földes-Papp Z, Wennmalm S, Rigler R (1999) Chem Phys 247:11–22
Engvall E, Perlmann P (1971) Immunochemistry 8:871–874
Hendrickson WA, Pahler A, Smith JL, Satow Y, Merritt EA, Phizackerley RP (1989) Proc Natl Acad Sci USA 86:2190–2194
Jacobson RH, Zhang XJ, DuBose RF, Matthews BW (1994) Nature 369:761–766
Kerkhof L (1992) Anal Biochem 205:359–364
Kitigawa T, Aikawa T (1976) J Biochem 79:233–236
Krylov SN, Dovichi NJ (2000) Anal Chem 72:111R–128R
Nichols ER, Craig DB (2008) Electrophoresis 29:4257–4269
O’Sullivan MJ, Gnemmi E, Morris D, Chieregatti G, Simmons M, Simmonds AD, Bridges JW, Marks V (1978) FEBS Lett 95:311–313
Shoemaker GK, Juers DH, Coombs JML, Matthews BW, Craig DB (2003) Biochemistry 42:1707–1710
Acknowledgments
This study was supported by a grant from the Natural Sciences and Engineering Research Council.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Craig, D.B., Henderson, A. Electrophoretic Heterogeneity Limits the Utility of Streptavidin-β-Galactosidase as a Probe in Free Zone Capillary Electrophoresis Separations. Protein J 32, 81–88 (2013). https://doi.org/10.1007/s10930-013-9464-7
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10930-013-9464-7