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Counteracting Effect of Charged Amino Acids Against the Destabilization of Proteins by Arginine

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Abstract

Studies on osmolyte-induced effects on proteins help in enhancing protein stability under stressed conditions for various applications. Using mixtures of osmolytes could indeed widen their applications. The combinatorial effects of osmolytes with methylamines are majorly found in the literature; however, such studies are limited on the amino acid class of osmolytes. The present study examines the effect of charged amino acids Arg, Asp, and Lys on the stability of RNase A and α-LA. The thermal stabilities of the proteins in the presence of osmolytes are monitored by absorption changes, and the structural changes are analyzed using fluorescence quenching and near-UV circular dichroism (CD). These results are compared with our previous report on the effect of Glu. Arg destabilizes both the proteins whereas Asp, Lys, and Glu stabilize the proteins. The extent of stability provided by Asp and Glu is almost same and higher than Lys in RNase A. However, the stability acquired in the presence of Asp and Lys is comparable for α-LA and Glu provides higher stability. Further, the quenching and CD results suggest that the addition of amino acids do not alter the structure of the proteins significantly. The counteracting abilities of the stabilizing amino acids (stAAs) against Arg are then investigated. The results show that Glu could counteract Arg at the lowest fraction in the mixture. Lys requires nearly equimolar concentration whereas Asp needs almost double the concentration to counteract Arg induced destabilization of the proteins. At higher concentrations, the counteracting ability of Asp and Lys is similar for both the proteins. The counteracting ratio might slightly vary among the proteins, and it is not necessary that the amino acid providing higher stability to the protein could more effectively counteract Arg. This could be due to the change in the extent of preferential hydration of the proteins by stAAs in the presence of Arg. The results suggest that the addition of stAAs could be an effective strategy to increase the protein stability in biotechnology and biopharma applications.

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Abbreviations

RNase A:

Bovine pancreatic ribonuclease A

α-LA:

Bovine alpha-lactalbumin

K SV :

Stern–Volmer constant

CD:

Circular dichroism

stAA:

Stabilizing amino acids

References

  1. Arakawa, T., Tsumoto, K., Kita, Y., Chang, B., & Ejima, D. (2007). Biotechnology applications of amino acids in protein purification and formulations. Amino Acids, 33(4), 587–605.

    Article  CAS  PubMed  Google Scholar 

  2. Tsumoto, K., Umetsu, M., Kumagai, I., Ejima, D., Philo, J. S., & Arakawa, T. (2004). Role of arginine in protein refolding, solubilization, and purification. Biotechnology Progress, 20(5), 1301–1308.

    Article  CAS  PubMed  Google Scholar 

  3. Bailey, T. L., Wang, M., Solocinski, J., Nathan, B. P., Chakraborty, N., & Menze, M. A. (2015). Protective effects of osmolytes in cryopreserving adherent neuroblastoma (Neuro-2a) cells. Cryobiology., 71(3), 472–480.

    Article  CAS  PubMed  Google Scholar 

  4. Hubalek, Z. (2003). Protectants used in the cryopreservation of microorganisms. Cryobiology., 46(3), 205–229.

    Article  CAS  PubMed  Google Scholar 

  5. Ishihara, T., & Hosono, M. (2015). Improving impurities clearance by amino acids addition to buffer solutions for chromatographic purifications of monoclonal antibodies. Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences, 995-996, 107–114.

    Article  CAS  PubMed  Google Scholar 

  6. Ejima, D., Yumioka, R., Tsumoto, K., & Arakawa, T. (2005). Effective elution of antibodies by arginine and arginine derivatives in affinity column chromatography. Analytical Biochemistry, 345(2), 250–257.

    Article  CAS  PubMed  Google Scholar 

  7. Shukla, D., Shinde, C., & Trout, B. L. (2009). The Journal of Physical Chemistry. B, 113, 12546–12554.

    Article  CAS  PubMed  Google Scholar 

  8. Prabhu, N., & Sharp, K. (2006). Protein−solvent interactions. Chemical Reviews, 106(5), 1616–1623.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Shah, D., & Shaikh, A. R. (2016). Interaction of arginine, lysine, and guanidine with surface residues of lysozyme: implication to protein stability. Journal of Biomolecular Structure & Dynamics, 34(1), 104–114.

    Article  CAS  Google Scholar 

  10. Bozorgmehr, M. R., & Monhemi, H. (2015). How can a free amino acid stabilize a protein? Insights from molecular dynamics simulation. Journal of Solution Chemistry, 44(1), 45–53.

    Article  CAS  Google Scholar 

  11. Golovanov, A. P., Hautbergue, G. M., Wilson, S. A., & Lian, L. Y. (2004). A simple method for improving protein solubility and long-term stability. Journal of the American Chemical Society, 126(29), 8933–8939.

    Article  CAS  PubMed  Google Scholar 

  12. Anumalla, B., & Prabhu, N. P. (2018). Glutamate induced thermal equilibrium intermediate and counteracting effect on chemical denaturation of proteins. The Journal of Physical Chemistry. B, 122(3), 1132–1144.

    Article  CAS  PubMed  Google Scholar 

  13. Tsumoto, K., Shinoki, K., Kondo, H., Uchikawa, M., Juji, T., & Kumagai, I. (1998). Highly efficient recovery of functional single-chain Fv fragments from inclusion bodies overexpressed in Escherichia coli by controlled introduction of oxidizing reagent—application to a human single-chain Fv fragment. Journal of Immunological Methods, 219(1-2), 119–129.

    Article  CAS  PubMed  Google Scholar 

  14. Buchner, J., Pastan, I., & Brinkmann, U. (1992). A method for increasing the yield of properly folded recombinant fusion proteins: single-chain immunotoxins from renaturation of bacterial inclusion bodies. Analytical Biochemistry, 205(2), 263–270.

    Article  CAS  PubMed  Google Scholar 

  15. Shiraki, K., Kudou, M., Fujiwara, S., Imanaka, T., & Takagi, M. (2002). Biophysical effect of amino acids on the prevention of protein aggregation. Journal of Biochemistry, 132(4), 591–595.

    Article  CAS  PubMed  Google Scholar 

  16. Tsumoto, K., Abe, R., Ejima, D., & Arakawa, T. (2010). Non-denaturing solubilization of inclusion bodies. Current Pharmaceutical Biotechnology, 11(3), 309–312.

    Article  CAS  PubMed  Google Scholar 

  17. Tsumoto, K., Umetsu, M., Kumagai, I., Ejima, D., & Arakawa, T. (2003). Solubilization of active green fluorescent protein from insoluble particles by guanidine and arginine. Biochemical and Biophysical Research Communications, 312(4), 1383–1386.

    Article  CAS  PubMed  Google Scholar 

  18. Startzel, P. (2018). Arginine as an excipient for protein freeze-drying: a mini review. Journal of Pharmaceutical Sciences, 107(4), 960–967.

    Article  CAS  PubMed  Google Scholar 

  19. Startzel, P., Gieseler, H., Gieseler, M., Abdul-Fattah, A. M., Adler, M., Mahler, H. C., & Goldbach, P. (2015). Freeze drying of l-arginine/sucrose-based protein formulations, part I: influence of formulation and arginine counter ion on the critical formulation temperature, product performance and protein stability. Journal of Pharmaceutical Sciences, 104(7), 2345–2358.

    Article  CAS  PubMed  Google Scholar 

  20. Startzel, P. Gieseler, H., Gieseler, M., Abdul-Fattah, A. M., Adler, M., Mahler, H-C. & Goldbach, P. (2016). Mannitol/L-Arginine-Based Formulation Systems for Freeze Drying of Protein Pharmaceuticals: Effect of the L-Arginine Counter Ion and Formulation Composition on the Formulation Properties and the Physical State of Mannitol. Journal of Pharmaceutical Sciences, 105(10), 3123–3135.

  21. Taneja, S., & Ahmad, F. (1994). Increased thermal stability of proteins in the presence of amino acids. The Biochemical Journal, 303(Pt 1), 147–153.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Ishibashi, M., Tsumoto, K., Tokunaga, M., Ejima, D., Kita, Y., & Arakawa, T. (2005). Is arginine a protein-denaturant? Protein Expression and Purification, 42(1), 1–6.

    Article  CAS  PubMed  Google Scholar 

  23. Arakawa, T., & Tsumoto, K. (2003). The effects of arginine on refolding of aggregated proteins: not facilitate refolding, but suppress aggregation. Biochemical and Biophysical Research Communications, 304(1), 148–152.

    Article  CAS  PubMed  Google Scholar 

  24. Yancey, P. H., Clark, M. E., Hand, S. C., Bowlus, R. D., & Somero, G. N. (1982). Living with water stress: evolution of osmolyte systems. Science., 217(4566), 1214–1222.

    Article  CAS  PubMed  Google Scholar 

  25. Xie, Q., Guo, T., Lu, J., & Zhou, H. M. (2004). The guanidine like effects of arginine on aminoacylase and salt-induced molten globule state. The International Journal of Biochemistry & Cell Biology, 36(2), 296–306.

    Article  CAS  Google Scholar 

  26. Hong, T., Iwashita, K., Handa, A., & Shiraki, K. (2017). Arginine prevents thermal aggregation of hen egg white proteins. Food Research International, 97, 272–279.

    Article  CAS  PubMed  Google Scholar 

  27. Eronina, T. B., Chebotareva, N. A., Sluchanko, N. N., Mikhaylova, V. V., Makeeva, V. F., Roman, S. G., Kleymenov, S. Y., & Kurganov, B. I. (2014). Dual effect of arginine on aggregation of phosphorylase kinase. International Journal of Biological Macromolecules, 68, 225–232.

    Article  CAS  PubMed  Google Scholar 

  28. Shah, D., Li, J., Shaikh, A. R., & Rajagopalan, R. (2012). Arginine-aromatic interactions and their effects on arginine-induced solubilization of aromatic solutes and suppression of protein aggregation. Biotechnology Progress, 28(1), 223–231.

    Article  CAS  PubMed  Google Scholar 

  29. Yoshizawa, S., Arakawa, T., & Shiraki, K. (2017). Thermal aggregation of human immunoglobulin G in arginine solutions: contrasting effects of stabilizers and destabilizers. International Journal of Biological Macromolecules, 104(Pt A), 650–655.

    Article  CAS  PubMed  Google Scholar 

  30. Yamaguchi, H., & Miyazaki, M. (2014). Refolding techniques for recovering biologically active recombinant proteins from inclusion bodies. Biomolecules., 4(1), 235–251.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Burgess, R. R. (2009). Chapter 17 refolding solubilized inclusion body proteins. Methods in Enzymology, 463, 259–282.

    Article  CAS  PubMed  Google Scholar 

  32. Rishi, V., Anjum, F., Ahmad, F., & Pfeil, W. (1998). Role of non-compatible osmolytes in the stabilization of proteins during heat stress. The Biochemical Journal, 329(Pt 1), 137–143.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Arakawa, T., & Timasheff, S. N. (1984). The mechanism of action of Na glutamate, lysine HCl, and piperazine-N,N'-bis(2-ethanesulfonic acid) in the stabilization of tubulin and microtubule formation. The Journal of Biological Chemistry, 259(8), 4979–4986.

    CAS  PubMed  Google Scholar 

  34. Bowlus, R. D., & Somero, G. N. (1979). Solute compatibility with enzyme function and structure: Rationales for the selection of osmotic agents and end-products of anaerobic metabolism in marine invertebrates. The Journal of Experimental Zoology, 208(2), 137–151.

    Article  CAS  PubMed  Google Scholar 

  35. Arakawa, T., Dix, D. B., & Chang, B. S. (2003). The effects of protein stabilizers on aggregation induced by multiple-stresses. Yakugaku Zasshi, 123(11), 957–961.

    Article  CAS  PubMed  Google Scholar 

  36. Jensen, W. A., Armstrong, J. M., De Giorgio, J., & Hearn, M. T. (1996). Stability studies on pig heart mitochondrial malate dehydrogenase: the effect of salts and amino acids. Biochimica et Biophysica Acta, 1296(1), 23–34.

    Article  PubMed  Google Scholar 

  37. Ou, W., Wang, R. S., Lu, J., & Zhou, H. M. (2002). The International Journal of Biochemistry & Cell Biology, 34, 970–982.

    Article  CAS  Google Scholar 

  38. Xie, Q., Guo, T., Wang, T., Lu, J., & Zhou, H. M. (2003). Aspartate-induced aminoacylase folding and forming of molten globule. The International Journal of Biochemistry & Cell Biology, 35(11), 1558–1572.

    Article  CAS  Google Scholar 

  39. Kheddo, P., Tracka, M., Armer, J., Dearman, R. J., Uddin, S., van der Walle, C. F., & Golovanov, A. P. (2014). The effect of arginine glutamate on the stability of monoclonal antibodies in solution. International Journal of Pharmaceutics, 473(1-2), 126–133.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Shukla, D., & Trout, B. L. (2011). The Journal of Physical Chemistry. B, 115, 11831–11839.

    Article  CAS  PubMed  Google Scholar 

  41. Kumar, N., & Kishore, N. (2014). The Journal of Chemical Thermodynamics, 78, 159–166.

    Article  CAS  Google Scholar 

  42. Poklar, N., Petrovcic, N., Oblak, M., & Vesnaver, G. (1999). Protein Science, 8, 832–840.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Agashe, V. R., & Udgaonkar, J. B. (1995). Thermodynamics of denaturation of barstar: evidence for cold denaturation and evaluation of the interaction with guanidine hydrochloride. Biochemistry., 34(10), 3286–3299.

    Article  CAS  PubMed  Google Scholar 

  44. Amdursky, N., & Stevens, M. M. (2015). Circular dichroism of amino acids: following the structural formation of phenylalanine. Chemphyschem., 16(13), 2768–2774.

    Article  CAS  PubMed  Google Scholar 

  45. Privalov, P. (1997). The Journal of Chemical Thermodynamics, 29, 447–474.

    Article  CAS  Google Scholar 

  46. Yancey, P. H. (2005). Organic osmolytes as compatible, metabolic and counteracting cytoprotectants in high osmolarity and other stresses. The Journal of Experimental Biology, 208(15), 2819–2830.

    Article  CAS  PubMed  Google Scholar 

  47. Burg, M. B., & Ferraris, J. D. (2008). Intracellular organic osmolytes: function and regulation. The Journal of Biological Chemistry, 283(12), 7309–7313.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Khan, S., Bano, Z., Singh, L. R., Hassan, M. I., Islam, A., & Ahmad, F. (2013). Testing the ability of non-methylamine osmolytes present in kidney cells to counteract the deleterious effects of urea on structure, stability and function of proteins. PLoS One, 8(9), e72533.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Sharma, S., Pathak, N., & Chattopadhyay, K. (2012). Journal of Proteins and Proteomics, 3, 129–139.

    CAS  Google Scholar 

  50. Xie, G., & Timasheff, S. N. (1997). Protein Science, 6, 211–221.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Xie, G., & Timasheff, S. N. (1997). The thermodynamic mechanism of protein stabilization by trehalose. Biophysical Chemistry, 64(1-3), 25–43.

    Article  CAS  PubMed  Google Scholar 

  52. Poddar, N. K., Ansari, Z. A., Singh, R. K., Moosavi-Movahedi, A. A., & Ahmad, F. (2008). Effect of monomeric and oligomeric sugar osmolytes on ΔGD, the Gibbs energy of stabilization of the protein at different pH values: Is the sum effect of monosaccharide individually additive in a mixture? Biophysical Chemistry, 138(3), 120–129.

    Article  CAS  PubMed  Google Scholar 

  53. Warepam, M., & Singh, L. R. (2015). Osmolyte mixtures have different effects than individual osmolytes on protein folding and functional activity. Archives of Biochemistry and Biophysics, 573, 77–83.

    Article  CAS  PubMed  Google Scholar 

  54. Lin, T. Y., & Timasheff, S. N. (1994). Why do some organisms use a urea-methylamine mixture as osmolyte? Thermodynamic compensation of urea and trimethylamine N-oxide interactions with protein. Biochemistry., 33(42), 12695–12701.

    Article  CAS  PubMed  Google Scholar 

  55. Singh, L. R., Ali Dar, T., Haque, I., Anjum, F., Moosavi-Movahedi, A. A., & Ahmad, F. (2007). Testing the paradigm that the denaturing effect of urea on protein stability is offset by methylamines at the physiological concentration ratio of 2:1 (urea:methylamines). Biochimica et Biophysica Acta, 1774(12), 1555–1562.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We acknowledge the instruments facilities supported by DST-FIST and UGC-SAP to the department.

Funding

This study is financially supported by the CSIR, India (01(2845)/16/EMR-II), and SERB, India (EMR/2016/003411).

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  1. Department of Biotechnology and Bioinformatics, School of Life Sciences, University of Hyderabad, Hyderabad, 500 046, India

    Bramhini Anumalla & N. Prakash Prabhu

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  1. Bramhini Anumalla
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Correspondence to N. Prakash Prabhu.

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Anumalla, B., Prabhu, N.P. Counteracting Effect of Charged Amino Acids Against the Destabilization of Proteins by Arginine. Appl Biochem Biotechnol 189, 541–555 (2019). https://doi.org/10.1007/s12010-019-03026-w

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