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Colorimetric Coupled Enzyme Assay for Cystathionine β-Synthase

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Abstract

A colorimetric coupled enzyme assay for the determination of cystathionine β-synthase activity is described. The method exploits cystathionine γ-lyase as an ancillary enzyme capable of transforming cystathionine, produced by cystathionine β-synthase, into cysteine. The cysteine is then spectrophotometrically detected at 560 nm, after its specific complexation with ninhydrin. This method was used to detect cystathionine β-synthase in crude extracts, and for the kinetic characterization of the enzyme partially purified from bovine kidney. A rapid two-step protocol is described for the partial purification of cystathionine γ-lyase from bovine kidney, aimed at a suitable and stable ancillary enzyme preparation.

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References

  1. R. Banerjee, R. Evande, O. Kabil, S. Ojha, and S. Taoka, Biochim. Biophys. Acta, 2003, 1647, 30.

    Article  CAS  PubMed  Google Scholar 

  2. S. M. Aitken, P. H. Lodha, and D. J. K. Morneau, Biochim. Biophys. Acta, 2011, 1814, 1511.

    Article  CAS  PubMed  Google Scholar 

  3. E. Mosharov, M. R. Cranford, and R. Banerjee, Biochemistry, 2000, 39, 13005.

    Article  CAS  PubMed  Google Scholar 

  4. P. W. Beatty and D. J. Reed, Arch. Biochem. Biophys., 1980, 204, 80.

    Article  CAS  PubMed  Google Scholar 

  5. V. Vitvitsky, S. Dayal, S. Stabler, Y. Zhou, H. Wang, S. R. Lentz, and R. Banerjee, Am. J. Physiol. Regul. Integr. Comp. Physiol., 2004, 287, R39.

    Article  CAS  PubMed  Google Scholar 

  6. J. P. Kraus, M. Janosik, V. Kozich, R. Mandell, V. Shih, M. P. Sperandeo, G. Sebastio, R. de Franchis, G. Andria, L. A. Kluijtmans, H. Blom, G. H. Boers, R. B. Gordon, P. Kamoun, M. Y. Tsai, W. D. Kruger, H. G. Koch, T. Ohura, and M. Gaustadnes, Hum. Mutat., 1999, 13, 362.

    Article  CAS  PubMed  Google Scholar 

  7. R. Clarke, L. Daly, K. Robinson, E. Naughten, S. Cahalane, B. Fowler, and I. Graham, N. Engl. J. Med., 1999, 324, 1149.

    Article  Google Scholar 

  8. K. N. Maclean, M. Gaustadnes, J. Oliveriusová, M. Janosîk, E. Kraus, V. Kozich, V. Kery, F. Skovby, N. Rüdiger, J. Ingerslev, S. P. Stabler, R. H. Allen, and J. P. Kraus, Hum. Mutat., 2002, 19, 641.

    Article  CAS  PubMed  Google Scholar 

  9. P. Whincup, H. Refsum, I. Perry, R. Morris, M. Walker, L. Lennon, A. Thomson, P. Ueland, and S. Ebrahim, Heart, 1999, 82, 448.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. R. Clarke, S. Lewington, A. Donald, C. Johnston, H. Refsum, I. Stratton, P. Jaques, M. M. Breteler, and R. Holman, J. Cardiovasc. Risk, 2001, 8, 363.

    Article  CAS  PubMed  Google Scholar 

  11. H. Kimura, N. Shibuya, and Y. Kimura, Antioxid. Redox Signal., 2012, 17, 45.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. V. Kery, G. Bukovska, and J. P. Kraus, J. Biol. Chem., 1994, 269, 25283.

    Article  CAS  PubMed  Google Scholar 

  13. E. W. Miles and J. P. Kraus, J. Biol. Chem., 2004, 279, 29871.

    Article  CAS  PubMed  Google Scholar 

  14. R. Banerjee and C. G. Zou, Arch. Biochem. Biophys., 2005, 433, 144.

    Article  CAS  PubMed  Google Scholar 

  15. S. H. Mudd, J. D. Finkelstein, F. Irreverre, and L. Laster, J. Biol. Chem., 1965, 240, 4382.

    Article  CAS  PubMed  Google Scholar 

  16. M. Suda, H. Nakagawa, and H. Kimura, Methods Enzymol., 1971, 17, 454.

    Article  Google Scholar 

  17. S. Taoka, S. Ohja, X. Shan, W. D. Kruger, and R. Banerjee, J. Biol. Chem., 1998, 273, 25179.

    Article  CAS  PubMed  Google Scholar 

  18. P. Manowitz, J. Racevskis, and D. G. Gilmour, Clin. Chim. Acta, 1970, 28, 269.

    Article  CAS  PubMed  Google Scholar 

  19. J. Kraus, S. Packman, B. Fowler, and L. E. Rosenberg, J. Biol. Chem., 1978, 253, 6523.

    Article  CAS  PubMed  Google Scholar 

  20. E. Borcsok and R. H. Abeles, Arch. Biochem. Biophys., 1982, 213, 695.

    Article  CAS  PubMed  Google Scholar 

  21. D. E. Smith, M. I. Mendes, L. A. Kluijtmans, M. C. Janssen, Y. M. Smulders, and H. J. Blom, J. Chromatogr. B, 2012, 911, 186.

    Article  CAS  Google Scholar 

  22. J. Krijt, J. Kopecká, A. Hnîzda, S. Moat, L. A. Kluijtmans, P. Mayne, and V. Kozich, J. Inherit. Metab. Dis., 2011, 34, 49.

    Article  CAS  PubMed  Google Scholar 

  23. T. G. Holt, B. K. Choi, N. S. Geoghagen, K. K. Jensen, Q. Luo, W. A. LaMarr, G. M. Makara, L. Malkowitz, C. C. Ozbal, Y. Xiong, C. Dufresne, and M. J. Luo, Assay Drug Dev. Technol., 2009, 7, 495.

    Article  CAS  PubMed  Google Scholar 

  24. P. Bronowicka-Adamska, J. Zagajewski, and M. Wróbel, Nitric Oxide, 2015, 46, 186.

    Article  CAS  PubMed  Google Scholar 

  25. S. Kashiwamata and D. M. Greenberg, Biochim. Biophys. Acta, 1970, 212, 488.

    Article  CAS  PubMed  Google Scholar 

  26. Y. Matsuo and D. M. Greenberg, J. Biol. Chem., 1958, 230, 545.

    Article  CAS  PubMed  Google Scholar 

  27. S. M. Aitken and J. F. S. Kirsch, Biochemistry, 2003, 42, 571.

    Article  CAS  PubMed  Google Scholar 

  28. W. Shen, M. K. McGath, R. Evande, and D. B. Berkowitz, Anal. Biochem., 2005, 342, 103.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. M. K. Gaitonde, Biochem. J., 1967, 104, 627.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. V. P. Shah, K. K. Midha, J. W. Findlay, H. M. Hill, J. D. Hulse, I. J. McGilveray, G. McKay, K. J. Miller, R. N. Patnaik, M. L. Powell, A. Tonelli, C. T. Viswanathan, and A. Yacobi, Pharm. Res., 2000, 17, 1551.

    Article  CAS  PubMed  Google Scholar 

  31. G. L. Ellman, Arch. Biochem. Biophys., 1959, 82, 70.

    Article  CAS  PubMed  Google Scholar 

  32. K. Heinonen, Biochem. J., 1973, 136, 1011.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. M. M. Bradford, Anal. Biochem., 1976, 72, 248.

    Article  CAS  PubMed  Google Scholar 

  34. R. Moschini, E. Peroni, R. Rotondo, G. Renzone, D. Melck, M. Cappiello, M. Srebot, E. Napolitano, A. Motta, A. Scaloni, U. Mura, and A. Del-Corso, Free Radie. Biol. Med., 2015, 83, 66.

    Article  CAS  Google Scholar 

  35. T. Chiku, D. Padovani, W. Zhu, S. Singh, V. Vitvitsky, and R. Banerjee, J. Biol. Chem., 2009, 284, 11601.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. M. S. Belew, F. I. Quazi, W. G. Willmore, and S. M. Aitken, Protein Expr. Purif., 2009, 64, 139.

    Article  CAS  PubMed  Google Scholar 

  37. N. Tudball and M. A. Reed, Biochem. Biophys. Res. Commun., 1975, 67, 550.

    Article  CAS  PubMed  Google Scholar 

  38. V. Kery, L. Poneleit, and J. P. Kraus, Arch. Biochem. Biophys., 1998, 355, 222.

    Article  CAS  PubMed  Google Scholar 

  39. M. Janosîk, V. Kery, M. Gaustadnes, K. N. Maclean, and J. P. Kraus, Biochemistry, 2001, 40, 10625

    Article  PubMed  Google Scholar 

  40. C. Steegborn, T. Clausen, P. Sondermann, U. Jacob, M. Worbs, S. Marinkovic, R. Huber, and M. C. Wahl, J. Biol. Chem., 1999, 274, 12675.

    Article  CAS  PubMed  Google Scholar 

  41. S. J. Stohs and D. Bagchi, Free Radic. Biol. Med., 1995, 78, 321.

    Article  Google Scholar 

  42. I. Cecconi, M. Moroni, P. G. Vilardo, M. Dal Monte, P. Borella, G. Rastelli, L. Costantino, D. Garland, D. Carper, J. M. Petrash, A. Del Corso, and U. Mura, Biochemistry, 1998, 37, 14167.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This work was supported in part by the Ministero Istruzione, Università, Ricerca, PRIN Project 2009M8FKBB_003 and in part by Pisa University. We are indebted to Dr. G. Pasqualetti and Dr. R. Di Sacco (veterinary staff of Consorzio Macelli S. Miniato, Pisa) for their valuable operation in the bovine kidney collection.

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Authors and Affiliations

  1. University of Pisa, Department of Biology, Biochemistry Unit, via S. Zeno, 51, 56123, Pisa, Italy

    Marco Rocchiccioli, Roberta Moschini, Laura Cappiello, Francesco Balestri, Mario Cappiello, Umberto Mura & Antonella Del-Corso

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  1. Marco Rocchiccioli
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  2. Roberta Moschini
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  3. Laura Cappiello
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  4. Francesco Balestri
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  7. Antonella Del-Corso
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Correspondence to Antonella Del-Corso.

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Rocchiccioli, M., Moschini, R., Cappiello, L. et al. Colorimetric Coupled Enzyme Assay for Cystathionine β-Synthase. ANAL. SCI. 32, 901–906 (2016). https://doi.org/10.2116/analsci.32.901

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  • DOI: https://doi.org/10.2116/analsci.32.901

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