Evidence for a potential-dependent reversible inactivation of urease adsorbed on a gold electrode

Abstract

The potentiometric collection mode of the scanning electrochemical microscope has been applied to a kinetic study of changes in immobilised enzyme activity. Urease adsorbs spontaneously onto gold surfaces from aqueous solution and the immobilised enzyme was shown to retain activity as a catalyst for the hydrolysis of urea to ammonium and bicarbonate ions. Urease activity was monitored using an ion-selective potentiometric tip electrode to detect a product (NH₄⁺) of the enzymatic reaction and the use of a 50 µm radius gold electrode to immobilise the enzyme results in a steady state diffusion field for the reaction products and enables quantitative interpretation of the data. The ammonium ion flux determined from the tip potential decreased by approximately 40% when the potential of the gold was scanned or stepped across the range from 0.0 to+0.4 V vs. Ag/AgCl. The decrease in flux is reversible and the original value almost completely recovered after the potential was returned to 0 V. The flux decrease is attributed to inactivation of the enzyme. The timescale of the inactivation is limited by the establishment of a steady state concentration profile of ammonium ions at the 50 µm radius source. The kinetics of the reactivation are significantly slower with an apparent first order rate constant of 0.022 s^-1. No faradaic processes were observed at the gold electrode over the potential range from 0.0 to +0.4 V. We therefore suggest that changes in the enzyme quaternary structure due to the changing electric field and ionic composition at the interface are responsible.

References

1. A. M. Klibanov, Science, 1983, 219, 722; G. G. Guilbault, Analytical uses of Immobilised Enzymes, in Modern Monographs in Analytical Chemistry, ed. G. G. Guilbault, Marcel Dekker, New York, 1984, vol. 2.
2. A. P. F. Turner, I. Karube and G. S. Wilson, Biosensors, Fundamentals and Applications, Oxford University Press, Oxford, 1987.
3. L. Hausling, H. Ringsdorf, F.-J. Schmitt and W. Knoll, Langmuir, 1991, 7, 1837.
4. J. H. Wang, L. W. Ruddock and A. E. G. Cass, Biosens. Bioelectron., 1994, 9, 647.
5. Th. Wink, S. J. van Zuilen, A. Bult and W. P. van Bennekom, Analyst, 1997, 122, 43R and references therein.
6. A. S. Ghatorae, G. Bell and P. J. Halling, Biotechnol. Bioeng., 1994, 43, 331; E. Katchalski, I. Silman and R. Goldman, Adv. Enzymol., 1971, 34, 445; K. M. R. Kallury, W. E. Lee and M. Thompson, Anal. Chem., 1993, 65, 2459.
7. E. L. Schmid, T. A. Keller, Z. Dienes and H. Vogel, Anal. Chem., 1997, 69, 1979; I. Willner, E. Katz, B. Willner, R. Blonder, V. Heleg-Shabatai and A. F. Buckmann, Biosens. Bioelectron., 1997, 12, 337; C. Kranz, T. Lotzbeyer, H.-L. Schmidt and W. Schuhmann, Biosens. Bioelectron., 1997, 12, 257.
8. A. L. Crumbliss, S. C. Perine, J. Stonehuerner, K. R. Tubergen, J. G. Zhao and R. W. Henkens, Biotechnol. Bioeng., 1992, 40, 483.
9. J. Hirst, A. Sucheta, B. A. C. Ackrell and F. A. Armstrong, J. Am. Chem. Soc., 1996, 118, 5031; K. S. V. Santhanam, N. Jespersen and A. J. Bard, J. Am. Chem. Soc., 1977, 99, 274.
10. K. L. Egodage, B. S. de Silva and G. S. Wilson, J. Am. Chem. Soc., 1997, 119, 5295.
11. K. L. Mclachlan and A. L. Crumbliss, Biotechnol. Bioeng., 1991, 37, 491.
12. B. R. Horrocks and A. J. Bard, unpublished data.
13. D. Ammann, Ion Selective Microelectrodes: Principles, Design and Application, Springer-Verlag, Berlin, 1986.
14. D. O. Wipf and A. J. Bard, J. Electrochem. Soc., 1991, 138, 469.
15. B. R. Horrocks, M. V. Mirkin, D. T. Pierce, A. J. Bard, K. Toth and G. Nagy, Anal. Chem., 1993, 65, 1213; E. Klusmann and J. W. Schultze, Electrochim. Acta, 1997, 42, 3123.
16. P. Vanysek, in CRC Handbook of Chemistry and Physics, ed. D. R. Lide, 73rd edn., CRC press, Boca Raton, FL, 1992.
17. J. Peterson, K. Harmon and C. Nieman, J. Biol. Chem., 1948, 176, 1.
18. A. S. Ghatorae, M. J. Guerra, G. Bell and P. J. Halling, Biotechnol. Bioeng., 1994, 44, 1355.
19. R. W. Lencki, J. Arul and R. J. Neufeld, Biotechnol. Bioeng., 1992, 40, 1421; 1427.
20. R. W. Lencki and R. J. Neufeld, Enzyme Microb. Technol., 1996, 19, 232.
21. C. C. Contaxis and F. J. Reithel, J. Biol. Chem., 1971, 246, 677.
22. S. Omar and M. Beauregard, J. Biotechnol., 1995, 39, 221.