Abstract
An effective, rapid and economical CE/LIF (capillary electrophoresis/laser-induced fluorescence) method was developed and applied to the characterization of signal peptidase (SPase) enzyme, which is a target for the screening of new drug candidates. In this method, CE separates the product from the substrate and LIF selectively detects the fluorescence-labeled product and substrate. By measuring the increase of the product as a function of time, one can monitor the progression of the enzyme reaction. The progression curves were also used for screening inhibitors for this enzyme. The effects of various reaction conditions were also studied and discussed. In addition, this CE/LIF method was applied to the determination of the enzyme activity, the quality control of the substrate and/or enzymes, and the cross-reactivity of inhibitors to the enzyme. It can be concluded that this method is suitable for high throughput screening (HTS) assays because it can deliver fast, sensitive, quantitative, and reliable results.
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References
A. J. M. Dricssen, P. Fekkes, and J. P. W. Van der Wlk, Curr. Opin. Microbiol, 1998, 1, 216.
A. Economou, Mol. Microbiol, 1998, 27, 27.
W. Wickner, A. J. M. Dricssen, and F. U. Hartl, Annu. Rev. Biochem., 1991, 60, 101.
W. R. Tschantz and R. E. Dalbey, “Methods in Enzymology”, 1994, Academic Press, Inc.
M. Takahara, D. W. Hibler, P. J. Barr, and J. A. Gerlt, J. Biol Chem., 1985, 260, 2670.
S. Chatterjee, D. Suciu, R. E. Dalbey, P. C. Kahn, and M. Inouye, J. Mol Biol, 1995, 245, 311.
M. Paetzel, R. E. Dalbey, and N. C. J. Strynadka, Pharmacol Ther., 2002, 87, 27.
L. J. Ma, X. Y. Gong, and E. S. Yeung, Anal Chem., 2000, 72, 3383.
G. Bruton, A. Huxley, P. O’Hanlon, B. Orlek, D. Eggleston, J. Humphries, S. Readshaw, A. West, S. Ashman, M. Brown, K. Moore, A. Pope, K. O’Dwyer, and L. Wang, Eur. J. Med. Chem., 2003, 38, 351.
F. M. Sung and R. E. Dalbey, J. Biol. Chem., 1992, 267, 13154.
J. L. Carlos, P. A. Klenotic, M. Paetzel, and N. C. Strynadka, Biochemistry, 2000, 39, 7276.
C. Van Valkenburgh, X. Chen, C. Mullins, H. Fang, and N. Green, J. Biol. Chem., 1999, 274, 11519.
P. A. Klenotic, J. L. Carlos, J. C. Samuelson, T. A. Schuenemann, and W. R. Tschantz, J. Biol. Chem., 2000, 275, 6490.
W. R. Tschantz, M. Sung, V. M. Delgado-Partin, and R. E. Dalbey, J. Biol. Chem., 1993, 268, 27349.
W. R. Tschantz, M. Paztel, G. Cao, D. Suciu, M. Inouye, and R. E. Dalbey, Biochemistry, 1995, 43, 3935.
R. L. Stein, M. D. F. S. Barbosa, and R. Bruckner, Biochemistry, 2000, 39, 7973.
A. E. Allsop, G. Brooks, G. Bruton, S. Coulton, P. D. Edwards, I. K. Hatton, A. C. Kaura, S. D. Mclean, N. D. Pearson, T. C. Smale, and R. Southgate, Bioorg. Med. Lett., 1995, 5, 443.
L. M. Levine, M. L. Michener, M. V. Toth, and B. C. Holwerda, Anal. Biochem., 1997, 247, 83.
I. K. Dey, P. H. Ray, and P. Novak, J. Biol. Chem., 1990, 265, 20069.
M. C. Roach, P. Gozel, and R. N. Zare, J. Chromatogr., 1988, 426, 129.
H. T. Chang and E. S. Yeung, Anal. Chem., 1995, 67, 1079.
W. T. Lowther and B. M. Dunn, Lett. Peptide Sci., 1994, 1, 89.
J. Tu, L. N. Anderson, J. Dai, and K. Peters, J. Chromatogr., B, 2003, 789, 323.
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Liu, DN., Li, L., Lu, WP. et al. Capillary Electrophoresis with Laser-Induced Fluorescence Detection as a Tool for Enzyme Characterization and Inhibitor Screening. ANAL. SCI. 24, 333–337 (2008). https://doi.org/10.2116/analsci.24.333
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DOI: https://doi.org/10.2116/analsci.24.333