Skip to main content
SpringerLink
Log in

Effects of pH on the kinetic reaction

  • Published:
Journal of the American Society for Mass Spectrometry

Abstract

In most cases, kinetic unfolding reactions of proteins follow a simple one-step mechanism that does not involve any detectable intermediates. One example for a more complicated unfolding reaction is the acid-induced denaturation of holo-myoglobin (hMb). This reaction proceeds through a transient intermediate and can be described by a sequential two-step mechanism (Konermann et al. Biochemistry 1997, 36, 6448–6454). Time-resolved electrospray ionization mass spectrometry (ESI MS) is a new technique for monitoring the kinetics of protein folding and unfolding in solution. Different protein conformations can be distinguished by the different charge state distributions that they generate during ESI. At the same time this technique allows monitoring the loss or binding of noncovalent protein ligands. In this work, time-resolved ESI MS is used to study the dependence of the kinetic unfolding mechanism of hMb on the specific solvent conditions used in the experiment. It is shown that hMb unfolds through a short-lived intermediate only at acidic pH. Under basic conditions no intermediate is observed. These findings are confirmed by the results of optical stopped-flow absorption experiments. This appears to be the first time that a dependence of the kinetic mechanism for protein unfolding on external conditions such as pH has been observed.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Anfinsen, C. B. Principles that govern the folding of protein chains. Science 1973, 181, 223–230.

    Article  CAS  Google Scholar 

  2. Theorell, H.; Åkesson, A. Studies on cytochrome c. II. The optical properties of pure cytochrome c and some of its derivatives. J. Am. Chem. Soc. 1941, 63, 1812–1820.

    Article  CAS  Google Scholar 

  3. Goto, Y.; Calciano, L. J.; Fink, A. Acid-induced folding of proteins. Proc. Natl. Acad. Sci. USA 1990, 87, 573–577.

    Article  CAS  Google Scholar 

  4. Moore, G. R.; Pettigrew, G. W. Cytochromes c: Evolutionary, Structural and Physicochemical Aspects; Springer: Heidelberg, 1990.

    Google Scholar 

  5. Dunbar, J.; Yennawat, H. P.; Banerjee, S.; Luo, J.; Farber, G. K. The effect of denaturants on protein structure. Protein Sci. 1997, 6, 1727–1733.

    Article  CAS  Google Scholar 

  6. Udgaonkar, J. B.; Baldwin, R. L. NMR evidence for an early framework intermediate on the folding pathway of ribonuclease A. Nature 1988, 335, 694–699.

    Article  CAS  Google Scholar 

  7. Roder, H.; Elöve, G. A.; Englander, S. W. Structural characterization of folding intermediates in cytochrome c by H-exchange labelling and proton NMR. Nature 1988, 335, 700–704.

    Article  CAS  Google Scholar 

  8. Dill, K. A.; Chan, H. S. From Levinthal to pathways to funnels. Natl. Struct. Biol. 1998, 4, 10–19.

    Article  Google Scholar 

  9. Creighton, T. E. Protein folding. Biochem. J. 1990, 270, 1–16.

    CAS  Google Scholar 

  10. Hughson, F. M.; Wright, P. E.; Baldwin, R. L. Structural characterisation of a partly folded apomyoglobin intermediate. Science 1990, 249, 1544–1548.

    Article  CAS  Google Scholar 

  11. Miranker, A.; Robinson, C. V.; Radford, S. E.; Aplin, R.; Dobson, C. M. Detection of transient protein folding populations by mass spectrometry. Science 1993, 262, 896–900.

    Article  CAS  Google Scholar 

  12. Tsui, V.; Garcia, C.; Cavagnero, S.; Siuzdak, G.; Dyson, H. J.; Wright, P. E. Quench-flow experiments combined with mass spectrometry show apomyoglobin folds through an obligatory intermediate. Protein Sci. 1999, 8, 45–49.

    Article  CAS  Google Scholar 

  13. Kiefhaber, T.; Labhardt, A. M.; Baldwin, R. L. Direct NMR evidence for an intermediate preceding the rate-limiting step in the unfolding of ribonuclease A. Nature 1995, 375, 513–515.

    Article  CAS  Google Scholar 

  14. Staniforth, R. A.; Bigotti, M. G.; Allocatelli, F. C. C.; Brunori, M. Unfolding of apomyoglobin from aplysia limacina: The effect of salst and pH on the cooperativity of folding. J. Mol. Biol. 1998, 275, 133–148.

    Article  CAS  Google Scholar 

  15. Panick, G.; Malessa, R.; Winter, R. Differences between the pressure- and temperature-induced denaturation and aggregation of beta-lactoglobulin A, B, and AB monitored by FT-IR spectroscopy and small-angle X-ray scattering. Biochemistry 1999, 38, 6512–6519.

    Article  CAS  Google Scholar 

  16. Sosnick, T. R.; Mayne, L.; Hiller, R.; Englander, S. W. The barriers in protein folding. Nature Struct. Biol. 1994, 1, 149–156.

    Article  CAS  Google Scholar 

  17. Konermann, L.; Rosell, F. I.; Mauk, A. G.; Douglas, D. J. Acid-induced denaturation of myoglobin studied by time-resolved electrospray ionization mass spectrometry. Biochemistry 1997, 36, 6448–6454.

    Article  CAS  Google Scholar 

  18. Konermann, L.; Collings, B. A.; Douglas, D. J. Cytochrome c folding kinetics studied by time-resolved electrospray ionization mass spectrometry. Biochemistry 1997, 36, 5554–5559.

    Article  CAS  Google Scholar 

  19. Zechel, D. L.; Konermann, L.; Withers, S. G.; Douglas, D. J. Pre-steady state kinetic analysis of an enzymatic reaction monitored by time-resolved electrospray ionization mass spectrometry. Biochemistry 1998, 37, 7664–7669.

    Article  CAS  Google Scholar 

  20. Chen, Y.-L.; Campbell, J. M.; Collings, B. A.; Konermann, L.; Douglas, D. J. Stability of a highly charged noncovalent complex in the gas phase: Holomyoglobin. Rapid Commun. Mass Spectrom. 1998, 12, 1003–1010.

    Article  CAS  Google Scholar 

  21. Lee, V. W. S.; Chen, Y.-L.; Konermann, L. Reconstitution of acid-denatured holo-myoglobin studied by time-resolved electrospray ionization mass spectrometry. Anal. Chem. 1999, 71, 4154–4159.

    Article  CAS  Google Scholar 

  22. Konermann, L. Monitoring reaction kinetics by continuous-flow methods: The effects of convection and molecular diffusion under laminar flow conditions. J. Phys. Chem. A 1999, 103, 7210–7216.

    Article  CAS  Google Scholar 

  23. Chowdhury, S. K.; Katta, V.; Chait, B. T. Probing conformational changes in proteins by mass spectrometry. J. Am. Chem. Soc. 1990, 112, 9012–9013.

    Article  CAS  Google Scholar 

  24. Katta, V.; Chait, B. T. Observation of the heme—globin complex in native myoglobin by electrospray-ionisation mass spectrometry. J. Am. Chem. Soc. 1991, 113, 8534–8535.

    Article  CAS  Google Scholar 

  25. Loo, J. A.; Edmonds, C. G.; Udseh, H. R.; Smith, R. D. Effect of reducing disulfide-containing proteins on electrospray ionisation mass spectra. Anal. Chem. 1990, 62, 693–698.

    Article  CAS  Google Scholar 

  26. Konermann, L.; Douglas, D. J. Equilibrium unfolding of proteins monitored by electrospray ionization mass spectrometry: Distinguishing two-state from multi-state transitions. Rapid Commun. Mass Spectrom. 1998, 12, 435–442.

    Article  CAS  Google Scholar 

  27. Konermann, L.; Douglas, D. J. Acid-induced unfolding of cytochrome c at different methanol concentrations: Electrospray ionization mass spectrometry specifically monitors changes in the tertiary structure. Biochemistry 1997, 36, 12296–12302.

    Article  CAS  Google Scholar 

  28. Wang, F.; Tang, X. Conformational heterogeneity and stability of apomyoglobin studied by hydrogen/deuterium exchange and electrospray ionization mass spectrometry. Biochemistry 1996, 35, 4069–4078.

    Article  CAS  Google Scholar 

  29. Vis, H.; Heinemann, U.; Dobson, C. M.; Robinson, C. V. Detection of a monomeric intermediate associated with dimerization of protein Hu by mass spectrometry. J. Am. Chem. Soc. 1998, 120, 6427–6428.

    Article  CAS  Google Scholar 

  30. Pan, X. M.; Sheng, X. R.; Zhou, J. M. Probing subtle acid-induced conformational changes of ribonuclease A by electrospray mass spectrometry. FEBS Lett. 1997, 402, 25–27.

    Article  CAS  Google Scholar 

  31. Loo, J. A.; Loo, R. R. O.; Light, K. J.; Edmonds, C. G.; Smith, R. D. Multiply charged negative ions by electrospray ionization of polypeptides and proteins. Anal. Chem. 1992, 64, 81–88.

    Article  CAS  Google Scholar 

  32. Veenstra, T. D.; Johnson, K. L.; Tomlinson, A. J.; Naylor, S.; Kumar, R. Determination of calcium-binding sites in rat brain calbindin D28 K by electrospray ionization mass spectrometry. Biochemistry 1997, 36, 3535–3542.

    Article  CAS  Google Scholar 

  33. Veenstra, T. D.; Johnson, K. L.; Tomlinson, A. J.; Craig, T. A.; Kumar, R.; Naylor, S. Zinc-induced conformational changes in the DNA-binding domain of the vitamin D receptor determined by electrospray ionization mass spectrometry. J. Am. Soc. Mass. Spectrom. 1998, 9, 8–14.

    Article  CAS  Google Scholar 

  34. Nemirovskiy, O. V.; Ramanathan, R.; Gross, M. L. Investigation of calcium-induced, noncovalent association of calmodulin with melittin by electrospray ionization mass spectrometry. J. Am. Soc. Mass Spectrom. 1997, 8, 809–812.

    Article  CAS  Google Scholar 

  35. Hu, P.; Loo, J. A. Determining calcium-binding stoichiometry and cooperativity of parvalbumin and calmodulin by mass spectrometry. J. Mass Spectrom. 1995, 30, 1076–1082.

    Article  CAS  Google Scholar 

  36. Trigo-Gonzalez, G.; Awang, G.; Racher, K.; Neden, K.; Borgford, T. Helix variants of troponin C with tailored calcium affinities. Biochemistry 1993, 32, 9826–9831.

    Article  CAS  Google Scholar 

  37. Feng, R.; Konishi, Y. Stepwise refolding of acid-denatured myoglobin: Evidence from electrospray mass spectrometry. J. Am. Soc. Mass Spectrom. 1993, 4, 638–645.

    Article  CAS  Google Scholar 

  38. Loo, J. A. Studying noncovalent protein complexes by electrospray ionization mass spectrometry. Mass Spectrom. Rev. 1997, 16, 1–23.

    Article  CAS  Google Scholar 

  39. Evans, S. V.; Brayer, G. D. High-resolution study of the three-dimensional structure of horse heart metmyoglobin. J. Mol. Biol. 1990, 213, 885–897.

    Article  CAS  Google Scholar 

  40. Konermann, L.; Douglas, D. J. Unfolding of proteins monitored by electrospray ionization mass spectrometry: A comparison of positive and negative ion modes. J. Am. Soc. Mass Spectrom. 1998, 9, 1248–1254.

    Article  CAS  Google Scholar 

  41. Mansoori, B. A.; Volmer, D. A.; Boyd, R. K. ‘Wrong-way-round’ electrospray ionization of amino acids. Rapid Commun. Mass Spectrom. 1997, 11, 1120–1130.

    Article  CAS  Google Scholar 

  42. Collings, B. A.; Douglas, D. J. Conformation of gas-phase myoglobin ions. J. Am. Chem. Soc. 1996, 118, 4488–4489.

    Article  CAS  Google Scholar 

  43. Hargrove, M. S.; Olson, J. S. The stability of holomyoglobin is determined by heme affinity. Biochemistry 1996, 35, 11310–11318.

    Article  CAS  Google Scholar 

  44. Shen, L. L.; Hermans, J. Kinetics of conformation change of sperm-whale myoglobin. I. Folding and unfolding of metmyoglobin following pH jump. Biochemistry 1972, 11, 1836–1841.

    Article  CAS  Google Scholar 

  45. Beechem, J. M.; Ameloot, M.; Brand, L. Global and target analysis of complex decay phenomena. Anal. Instrum. 1985, 14, 379–402.

    Article  CAS  Google Scholar 

  46. Le Blanc, J. C. Y.; Guevremont, R.; Siu, K. W. M. Electrospray mass spectrometry of some proteins and the aqueous solution acid/base equilibrium model in the negative ion detection mode. Int. J. Ion. Mass Spectrom. Ion Processes 1993, 125, 145–153.

    Article  Google Scholar 

  47. Guevremont, R.; Siu, K. W. M.; Le Blanc, J. C. Y.; Berman, S. S. Are the electrospray mass spectra of proteins related to their aqueous solution chemistry? J. Am. Soc. Mass Spectrom. 1992, 3, 216–224.

    Article  CAS  Google Scholar 

  48. Laidler, K. J.; Meiser, J. H. Physical Chemistry, 3rd ed.; Houghton Mifflin Company: Boston, 1999.

    Google Scholar 

  49. Sage, J. T.; Morikis, D.; Champion, P. M. Spectroscopic studies of myoglobin at low pH: Heme structure and ligation. Biochemistry 1991, 30, 1227–1237.

    Article  CAS  Google Scholar 

  50. Brandts, J. F.; Brennan, M.; Lin, L. Unfolding and refolding occur much faster for a proline-free protein than for most proline-containing proteins. Proc. Natl. Acad. Sci. USA 1977, 74, 4178–4181.

    Article  CAS  Google Scholar 

  51. Bychkova, V. E.; Dujsekina, A. E.; Klenin, S. I.; Tiktopulo, E. I.; Uversky, V. N.; Ptitsyn, O. B. Molten globule-like state of cytochrome c under conditions simulating those near the membrane surface. Biochemistry 1996, 35, 6058–6063.

    Article  CAS  Google Scholar 

  52. La Mar, G. N.; Davis, N. L.; Parish, D. W.; Smith, K. M. Heme orientational disorder in reconstitute and native sperm whale myoglobin. J. Mol. Biol. 1983, 168, 887–896.

    Article  Google Scholar 

  53. La Mar, G. N.; Toi, H.; Krishnamoorthi, R. Proton NMR investigation of the rate and mechanism of heme rotation in sperm whale myoglobin: Evidence for intramolecular reorientation about a heme twofold axis. J. Am. Chem. Soc. 1984, 106, 6395–6401.

    Article  Google Scholar 

  54. Adams, P. A. The kinetics and mechanism of the recombination reaction between apomyoglobin and haemin. Biochem. J. 1976, 159, 371–376.

    CAS  Google Scholar 

  55. Adams, P. A. The kinetics of the recombination reaction between apomyoglobin and alkaline haematin. Biochem. J. 1977, 163, 153–158.

    CAS  Google Scholar 

  56. Hunter, C. H.; Mauk, A. G.; Douglas, D. J. Dissociation of heme from myoglobin and cytochrome b5: Comparison of behavior in solution and the gas phase. Biochemistry 1997, 36, 1018–1025.

    Article  CAS  Google Scholar 

Download references

Author information

Author notes

  1. Oyebola O. Sogbein

    Present address: McMaster University, 1280 Main Street W., Mail Box #92, L8S 4M3, Hamilton, ON, Canada

Authors and Affiliations

  1. Department of Chemistry, The University of Western Ontario, N6A 5B7, London, ON, Canada

    Oyebola O. Sogbein, Douglas A. Simmons & Lars Konermann

Authors
  1. Oyebola O. Sogbein
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Douglas A. Simmons
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Lars Konermann
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Lars Konermann.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sogbein, O.O., Simmons, D.A. & Konermann, L. Effects of pH on the kinetic reaction. J Am Soc Mass Spectrom 11, 312–319 (2000). https://doi.org/10.1016/S1044-0305(99)00149-X

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1016/S1044-0305(99)00149-X

Keywords

Search

Navigation