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Stabilization Studies on Bacterially Produced Human Paraoxonase 1 for Improving Its Shelf Life

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Abstract

Human paraoxonase 1 (h-PON1) is a ~40 kDa multi-tasking enzyme that plays a major role in determining individual susceptibility towards various disease conditions. It is a strong candidate for the development of therapeutic intervention for various diseases and other conditions in humans. However, purified h-PON1 is unstable and there is a need to find condition(s) that can increase the shelf life of the enzyme. In this report, we present the results of our investigation on the effect of excipients on the stability of bacterially produced human PON1 when stored under different storage conditions. Our results show that (a) glycine and serine are most effective in stabilizing the enzyme when stored in aqueous buffer at 25 °C for 30 days, and (b) trehalose, maltose, and BSA exerted maximum stabilization effect when the enzyme was stored in freeze-dried form at 25 °C for 60 days. Results of this study can be used to increase the shelf life of purified h-PON1 enzyme.

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Notes

  1. P. Bajaj, G. Aggarwal, R.K. Tripathy, and A.H. Pande, Interplay between amino acid residues at positions 192 and 115 in modulating hydrolytic activities of human paraoxonase 1. (submitted for publication).

Abbreviations

h-PON1:

Human paraoxonase 1

bph-PON1:

Bacterially produced human paraoxonase 1

CMC:

Carboxy methyl cellulose

PAA:

Polyacrylic acid

References

  1. La Du BN, Aviram M, Billecke S, Navab M, Primo-Parmo S, Sorenson RC, Standiford TJ (1998) On the physiological role(s) of the paraoxonases. Chem Biol Interact, 119–120, 379–388

  2. Ahmed, Z., Ravandi, A., Maguire, G. F., Emili, A., Draganov, D., La Du, B. N., Kuksis, A., & Connelly, P. W. (2002). Multiple substrates for paraoxonase-1 during oxidation of phosphatidylcholine by peroxynitrite. Biochem Biophys Res Commun, 290, 391–396.

    Article  CAS  Google Scholar 

  3. Mackness, B., & Mackness, M. (2010). Anti-inflammatory properties of paraoxonase-1 in atherosclerosis. Adv Exp Med Biol, 660, 143–151.

    Article  CAS  Google Scholar 

  4. Shih, D. M., Gu, L., Xia, Y. R., Navab, M., Li, W. F., Hama, S., Castellani, L. W., Furlong, C. E., Costa, L. G., & Fogelman, A. M. (1998). Mice lacking serum paraoxonase are susceptible to organophosphate toxicity and atherosclerosis. Nature, 394, 284–287.

    Article  CAS  Google Scholar 

  5. Koren-Gluzer, M., Aviram, M., Meilin, E., & Hayek, T. (2011). The antioxidant HDL-associated paraoxonase-1 (PON1) attenuates diabetes development and stimulates beta-cell insulin release. Atherosclerosis, 219, 510–518.

    Article  CAS  Google Scholar 

  6. Li, W. F., Furlong, C. E., & Costa, L. G. (1995). Paraoxonase protects against chlorpyrifos toxicity in mice. Toxicol Lett, 76, 219–226.

    Article  CAS  Google Scholar 

  7. Camps, J., Pujol, I., Ballester, F., Joven, J., & Simo, J. M. (2011). Paraoxonases as potential antibiofilm agents: their relationship with quorum-sensing signals in gram-negative bacteria. Antimicrob Agents Chemother, 55, 1325–1331.

    Article  CAS  Google Scholar 

  8. Chambers, J. E. (2008). PON1 multitasks to protect health. Proc Natl Acad Sci, 105, 12639–12640.

    Article  CAS  Google Scholar 

  9. Tavori, H., Vaya, J., & Aviram, M. (2010). Paraoxonase 1 attenuates human plaque atherogenicity: relevance to the enzyme lactonase activity. Adv Exp Med Biol, 660, 99–111.

    Article  CAS  Google Scholar 

  10. Lenz, D. E., Yeung, D., Smith, J. R., Sweeney, R. E., Lumley, L. A., & Cerasoli, D. M. (2007). Stoichiometric and catalytic scavengers as protection against nerve agent toxicity: a mini review. Toxicology, 233, 31–39.

    Article  CAS  Google Scholar 

  11. Rochu, D., Chabriere, E., & Masson, P. (2007). Human paraoxonase: a promising approach for pre-treatment and therapy of organophosphorus poisoning. Toxicology, 233, 47–59.

    Article  CAS  Google Scholar 

  12. Ritcher, R. J., Jarvik, G. P., & Furlong, C. E. (2010). Paraoxonase 1 status as a risk factor for disease or exposure. Adv Exp Med Biol, 660, 29–35.

    Article  Google Scholar 

  13. Gugliucci, A. (2010). Beyond the antioxidant properties: pomegranate juice polyphenols increase hepatocyte paraoxonase 1 secretion. Atherosclerosis, 208, 28–29.

    Article  CAS  Google Scholar 

  14. Gan, K. N., Smolen, A., Eckerson, H. W., & La Du, B. N. (1991). Purification of human serum paraoxonase/arylesterase: evidence for one esterase catalyzing both activities. Drug Metab Dispos, 19, 100–106.

    CAS  Google Scholar 

  15. Golmanesh, L., Mehrani, H., & Tabei, M. (2008). Simple procedures for purification and stabilization of human paraoxonase 1. J Biochem Biophys Methods, 70, 1037–1042.

    Article  CAS  Google Scholar 

  16. Rochu, D., Renault, F., Cléry-Barraud, C., Chabrière, E., & Masson, P. (2007). Stability of highly purified human paraoxonase (PON1): association with human phosphate binding protein (HPBP) is essential for preserving its active conformation(s). Biochim Biophys Acta, 1774, 874–883.

    Article  CAS  Google Scholar 

  17. Nguyen, S. D., & Sok, D. E. (2003). Beneficial effect of oleoylated lipids on paraoxonase 1: protection against oxidative inactivation and stabilization. Biochem J, 375, 275–285.

    Article  CAS  Google Scholar 

  18. Otto, T. C., Kasten, S. A., Kovaleva, E., Liu, Z., Buchman, G., Tolosa, M., Davis, D., Smith, J. R., Balcerzak, R., & Lenz, D. E. (2010). Purification and characterization of functional human paraoxonase-1 expressed in Trichoplusia ni larvae. Chem Biol Interact, 187, 388–392.

    Article  CAS  Google Scholar 

  19. Draganov, D. I., Teiber, J. F., Speelman, A., Osawa, Y., Sunahara, R., & La Du, B. N. (2005). Human paraoxonases (PON1, PON2, and PON3) are lactonases with overlapping and distinct substrate specificities. J Lipid Res, 46, 1239–1247.

    Article  CAS  Google Scholar 

  20. Manning, M. C., Chou, D. K., Murphy, B. M., Payne, R. W., & Katayama, D. S. (2010). Stability of protein pharmaceuticals: an update. Pharm Res, 27, 544–575.

    Article  Google Scholar 

  21. Jeong, S. H. (2010). Analytical methods and formulation factors to enhance protein stability in solution. Arch Pharm Res, 35, 1871–1886.

    Article  Google Scholar 

  22. Ohtake, S., Kita, Y., & Arakawa, T. (2011). Interactions of formulation excipients with proteins in solution and in the dried state. Adv Drug Deliv Rev, 63, 1053–1073.

    Article  CAS  Google Scholar 

  23. Arakawa, T., Tsumoto, K., Kita, Y., Chang, B., & Ejima, D. (2007). Biotechnology applications of amino acids in protein purification and formulations. Amino Acids, 33, 587–605.

    Article  CAS  Google Scholar 

  24. Timasheff, S. N. (2002). Protein-solvent preferential interactions, protein hydration, and the modulation of biochemical reactions by solvent components. Proc Natl Acad Sci, 99, 9721–9726.

    Article  CAS  Google Scholar 

  25. Timasheff, S. N. (1995). Solvent stabilization of protein structure. Methods Mol Biol, 40, 253–269.

    CAS  Google Scholar 

  26. Arakawa, T., Pretrelskia, S. J., Kenneya, W. C., & Carpenter, J. F. (2001). Factors affecting short-term and long-term stabilities of proteins. Adv Drug Deliv Rev, 46, 307–326.

    Article  CAS  Google Scholar 

  27. Simpson, R. J. (2010). Stabilization of proteins for storage. Cold Spring Harbor Protocol. doi:10.1101/pdb.top79.

    Google Scholar 

  28. Bhambhani, A., Kissman, J. M., Joshi, S. B., Volkin, D. B., Kashi, R. S., & Middaugh, C. R. (2012). Formulation design and high-throughput excipient selection based on structural integrity and conformational stability of dilute and highly concentrated IGg1 monoclonal antibody solutions. J Pharm Sci, 101, 1120–1135.

    Article  CAS  Google Scholar 

  29. Mueller, M., Loh, M. Q. T., Tee, D. H. Y., Yang, Y., & Jungbauer, A. (2013). Liquid formulations for long-term storage of monoclonal IgGs. Appl Biochem Biotechnol, 169, 1431–1448.

    Article  CAS  Google Scholar 

  30. Hasset, K. J., Cousins, M. C., Rabia, L. A., Chadwick, C. M., Ohara, J. M., Nandi, P., Brey, R. N., Mantis, N. J., Carpenter, J. F., & Randolf, T. W. (2013). Stabilization of a recombinant ricin toxin A subunit vaccine through lyophilisation. Eur J Pharm Biopharm, 85, 279–286.

    Article  Google Scholar 

  31. Sambrook, J., Fritsch, E. F., & Maniatis, T. (2001). Molecular cloning: a laboratory manual (2nd ed.). Cold Spring Harbor: Cold Spring Harbor Laboratory Press.

    Google Scholar 

  32. Middelberg, A. P. (2001). Preparative protein refolding. Trends Biotechnol, 20, 437–443.

    Article  Google Scholar 

  33. Kar, S., Patel, M. A., Tripathy, R. K., Bajaj, P., Suvarnakar, U. V., & Pande, A. H. (2012). Oxidized phospholipid content destabilizes the structure of reconstituted high density lipoprotein particles and changes their function. Biochim Biophys Acta, 1821, 1200–1210.

    Article  CAS  Google Scholar 

  34. Chi, E. Y., Krishnan, S., Randolph, T. W., & Carpenter, J. F. (2003). Physical stability of proteins in aqueous solution: mechanism and driving forces in nonnative protein aggregation. Pharm Res, 20, 1325–1336.

    Article  CAS  Google Scholar 

  35. Carpenter, J. F., Pikal, M. J., Byeong, S., & Randolph, T. W. (1997). Rational design of stable lyophilized protein formulations: some practical advice. Pharm Res, 14, 969–975.

    Article  CAS  Google Scholar 

  36. Arakawa, T., Ejima, D., Tsumoto, K., Obeyama, N., Tanaka, Y., Kita, Y., & Timshaff, S. N. (2007). Suppression of protein interactions by arginine: a proposed mechanism of the arginine effects. Biophys Chem, 127, 1–8.

    Article  CAS  Google Scholar 

  37. Arakawa, T., Carpenter, J. F., Kita, Y. A., & Crowe, J. H. (1990). The basis for toxicity of certain cryoprotectants: a hypothesis. Cryobiology, 27, 401–415.

    Article  CAS  Google Scholar 

  38. Franks, F., & Hatley, R. H. M. (1991). Stability of proteins at subzero temperatures: thermodynamics and some ecological consequences. Pure Appl Chem, 63, 1367–1380.

    Article  CAS  Google Scholar 

  39. Hatley, R. H. M., & Franks, F. (1986). Denaturation of lactate dehydrogenase at subzero temperatures. Cryo-Letters, 7, 226–233.

    CAS  Google Scholar 

  40. Chang, B. S., Kendrick, B. S., & Carpenter, J. E. (1996). Surface-induced denaturation of proteins during freezing and its inhibition by surfactants. J Pharm Sci, 85, 1325–1330.

    Article  CAS  Google Scholar 

  41. Fields, G. B., Alonso, D. O. V., Stigter, D., & Dill, K. A. (1992). Theory for the aggregation of proteins and copolymers. J Phys Chem, 96, 3974–3981.

    Article  CAS  Google Scholar 

  42. Privalov, P. L. (1990). Cold denaturation of proteins. Crit Rev Biochem Mol Biol, 25, 281–305.

    Article  CAS  Google Scholar 

  43. Bock, P. E., & Frieden, C. (1978). Another look at the cold lability of enzymes. Trends Biochem Sci, 3, 100–103.

    Article  CAS  Google Scholar 

  44. Ondrias, M. R., Rousseau, D. L., & Simon, S. R. (1981). Structural changes at the heme induced by freezing. Science, 213, 559–657.

    Article  Google Scholar 

  45. Carpenter, J. F., Crowe, L. M., & Crowe, J. H. (1987). Stabilization of phosphofructokinase with sugars during freeze-drying: characterization of enhanced protection in presence of divalent cations. Biochim Biophys Acta, 923, 109–115.

    Article  CAS  Google Scholar 

  46. Carpenter, J. F., Martin, B., Crowe, L. M., & Crowe, J. H. (1987). Stabilization of phosphofructokinase during air-drying with sugars and sugar/transition metal mixtures. Cryobiology, 24, 455–464.

    Article  CAS  Google Scholar 

  47. Crowe, J. H., Carpenter, J. F., Crowe, L. M., & Anchordoguy, T. J. (1990). Are freezing and dehydration similar stress vectore? A comparison of modes of interaction stabilizing solutes with biomolecules. Cryobiology, 27, 219–231.

    Article  CAS  Google Scholar 

  48. Schachman, H. K., & Lauffer, M. A. (1949). The hydration, size and shape of tobacco mosaic virus. J Am Chem Soc, 71, 536–541.

    Article  CAS  Google Scholar 

  49. Carpenter, J. F., Prestrelski, S. J., Anchrodoguy, T. J., & Arakawa, T. (1994). Interactions of stabilizers with proteins during freezing and drying, in formulation and delivery of proteins and peptides (pp. 134–147). Washington DC: ACS Symposium Series; American Chemical Society.

    Book  Google Scholar 

  50. Crowe, L. M., Reid, D. S., & Crowe, J. H. (1996). Is trehalose special for preserving dry biomaterials? Biophys J, 71, 2087–2093.

    Article  CAS  Google Scholar 

  51. Jain, N. K., & Roy, I. (2009). Effect of trehalose on protein structure. Protein Sci, 18, 24–36.

    CAS  Google Scholar 

  52. Chang, L., Shepherd, D., Sun, J., Ouellette, D., Grant, K. L., Tang, X., & Pikal, M. J. (2005). Mechanism of protein stabilization by sugars during freeze-drying and storage: native structure preservation, specific interaction, and/or immobilization in a glassy matrix? J Pharm Sci, 94, 1427–1444.

    Article  CAS  Google Scholar 

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Acknowledgments

This work was supported by the research grants to AHP from NIPER, SAS Nagar. The authors thank Prof. K.P.R. Kartha, Department of Chemistry, NIPER-SAS Nagar, for his valuable assistance in writing the manuscript. Priyanka Bajaj (CSIR-SPM-SRF) is thankful to CSIR, New Delhi, for financial support in the form of CSIR Fellowship.

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Correspondence to Abhay H. Pande.

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Bajaj, P., Pande, A.H. Stabilization Studies on Bacterially Produced Human Paraoxonase 1 for Improving Its Shelf Life. Appl Biochem Biotechnol 172, 3798–3809 (2014). https://doi.org/10.1007/s12010-014-0806-5

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