Skip to main content
SpringerLink
Log in

Reversible two-step unfolding of heme–human serum albumin: a 1H-NMR relaxometric and circular dichroism study

  • Original Paper
  • Published:
JBIC Journal of Biological Inorganic Chemistry Aims and scope Submit manuscript

Abstract

Human serum albumin (HSA) participates in heme scavenging, the bound heme turning out to be a reactivity center and a powerful spectroscopic probe. Here, the reversible unfolding of heme–HSA has been investigated by 1H-NMR relaxometry, circular dichroism, and absorption spectroscopy. In the presence of 6 equiv of myristate (thus fully saturating all available fatty acid binding sites in serum heme–albumin), 1.0 M guanidinium chloride induces some unfolding of heme–HSA, leading to the formation of a folding intermediate; this species is characterized by increased relaxivity and enhanced dichroism signal in the Soret region, suggesting a more compact heme pocket conformation. Heme binds to the folding intermediate with K d = (1.2 ± 0.1) × 10−6 M. In the absence of myristate, the conformation of the folding intermediate state is destabilized and heme binding is weakened [K d = (3.4 ± 0.1) × 10−5 M]. Further addition of guanidinium chloride (up to 5 M) brings about the usual denaturation process. In conclusion, myristate protects HSA from unfolding, stabilizing a folding intermediate state in equilibrium with the native and the fully unfolded protein, envisaging a two-step unfolding pathway for heme–HSA in the presence of myristate.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

B:

Basic

CD:

Circular dichroism

F:

Fast migrating

FA:

Fatty acid

GnCl:

Guanidinium chloride

HSA:

Human serum albumin

N:

Neutral

References

  1. Carter DC, Ho JX (1994) Adv Protein Chem 45:153–203

    Article  PubMed  CAS  Google Scholar 

  2. Peters T Jr (1996) All about albumin: biochemistry, genetics and medical applications. Academic Press, Orlando

    Google Scholar 

  3. Curry S (2002) Vox Sang 83:315–319

    PubMed  CAS  Google Scholar 

  4. Fasano M, Curry S, Terreno E, Galliano M, Fanali G, Narciso P, Notari S, Ascenzi P (2005) IUBMB Life 57:787–796

    Article  PubMed  CAS  Google Scholar 

  5. Ascenzi P, Bocedi A, Notari S, Fanali G, Fesce R, Fasano M (2006) Mini Rev Med Chem 6:483–489

    Article  PubMed  CAS  Google Scholar 

  6. Spector AA (1975) J Lipid Res 16:165–179

    PubMed  CAS  Google Scholar 

  7. Hamilton JA (2004) Prog Lipid Res 43:177–199

    Article  PubMed  CAS  Google Scholar 

  8. Simard JR, Zunszain PA, Ha CE, Yang JS, Bhagavan NV, Petitpas I, Curry S, Hamilton JA (2005) Proc Natl Acad Sci USA 102:17958–17963

    Article  PubMed  CAS  Google Scholar 

  9. Kragh-Hansen U, Watanabe H, Nakajou K, Iwao Y, Otagiri M (2006) J Mol Biol 363:702–712

    Article  PubMed  CAS  Google Scholar 

  10. Simard JR, Zunszain PA, Hamilton JA, Curry S (2006) J Mol Biol 361:336–351

    Article  PubMed  CAS  Google Scholar 

  11. Fasano M, Baroni S, Vannini A, Ascenzi P, Aime S (2001) J Biol Inorg Chem 6:650–658

    Article  PubMed  CAS  Google Scholar 

  12. Wardell MZ, Wang JX, Ho J, Robert J, Rüker F, Ruble J, Carter DC (2002) Biochem Biophys Res Commun 291:813–819

    Article  PubMed  CAS  Google Scholar 

  13. Zunszain PA, Ghuman J, Komatsu T, Tsuchida E, Curry S (2003) Struct Biol 3:6

    Article  Google Scholar 

  14. Fasano M, Fanali G, Leboffe L, Ascenzi P (2007) IUBMB Life 59:436–440

    Article  PubMed  CAS  Google Scholar 

  15. Fasano M, Fanali G, Fesce R, Ascenzi P (2008) In: Bolognesi M, di Prisco G, Verde C (eds) Dioxygen binding and sensing proteins. Springer, Heidelberg, pp 121–131

  16. Baroni S, Mattu M, Vannini A, Cipollone R, Aime S, Ascenzi P, Fasano M (2001) Eur J Biochem 268:6214–6220

    Article  PubMed  CAS  Google Scholar 

  17. Mattu M, Vannini A, Coletta M, Fasano M, Ascenzi P (2001) J Inorg Biochem 84:293–296

    Article  PubMed  CAS  Google Scholar 

  18. Fasano M, Mattu M, Coletta M, Ascenzi P (2002) J Inorg Biochem 91:487–490

    Article  PubMed  CAS  Google Scholar 

  19. Monzani E, Curto M, Galliano M, Minchiotti L, Aime S, Baroni S, Fasano M, Amoresano A, Salzano AM, Pucci P, Casella L (2002) Biophys J 83:2248–2258

    Article  PubMed  CAS  Google Scholar 

  20. Fanali G, Fesce R, Agrati C, Ascenzi P, Fasano M (2005) FEBS J 272:4672–4683

    Article  PubMed  CAS  Google Scholar 

  21. Fanali G, Bocedi A, Ascenzi P, Fasano M (2007) FEBS J 274:4491–4502

    Article  PubMed  CAS  Google Scholar 

  22. Wyman J Jr (1964) Adv Protein Chem 19:223–286

    Article  PubMed  CAS  Google Scholar 

  23. Yamasaki K, Maruyama T, Yoshimoto K, Tsutsumi Y, Narazaki R, Fukuhara A, Kragh-Hansen U, Otagiri M (1999) Biochim Biophys Acta 1432:313–323

    PubMed  CAS  Google Scholar 

  24. Ascenzi P, Bocedi A, Notari S, Menegatti E, Fasano M (2005) Biochem Biophys Res Commun 334:481–486

    Article  PubMed  CAS  Google Scholar 

  25. Dill K, Alonso DOV, Hutchinson K (1989) Biochemistry 28:5439–5449

    Article  PubMed  CAS  Google Scholar 

  26. Kosa T, Maruyama T, Sakai N, Yonemura N, Yahara S, Otagiri M (1998) Pharm Res 15:592–598

    Article  PubMed  CAS  Google Scholar 

  27. Farruggia B, Rodriguez F, Rigatuso R, Fidelio G, Picò G (2001) J Protein Chem 20:81–89

    Article  PubMed  CAS  Google Scholar 

  28. Santra MK, Banerjee A, Krishnakumar SS, Rahaman O, Panda D (2004) Eur J Biochem 271:1789–1797

    Article  PubMed  CAS  Google Scholar 

  29. Santra MK, Banerjee A, Rahaman O, Panda D (2005) Int J Biol Macromol 37:200–204

    Article  PubMed  CAS  Google Scholar 

  30. Ahmad B, Ankita, Khan RH (2005) Arch Biochem Biophys 437:159–167

    Article  PubMed  CAS  Google Scholar 

  31. Rezaei-Tavirani M, Moghaddamnia SH, Ranjbar B, Amani M, Marashi SA (2006) J Biochem Mol Biol 39:530–536

    CAS  Google Scholar 

  32. Fanali G, Ascenzi P, Fasano M (2007) Biophys Chem 129:29–35

    Article  PubMed  CAS  Google Scholar 

  33. Bradford MM (1976) Anal Biochem 72:248–254

    Article  PubMed  CAS  Google Scholar 

  34. Boffi A, Das TK, Della Longa S, Spagnuolo C, Rousseau DL (1999) Biophys J 77:1143–1149

    Article  PubMed  CAS  Google Scholar 

  35. Koenig SH, Brown RDIII (1990) Progr NMR Spectrosc 22:487–567

    Article  CAS  Google Scholar 

  36. Bertini I, Luchinat C (1986) NMR of paramagnetic molecules in biological systems. Benjamin/Cummings, Menlo Park

    Google Scholar 

  37. Banci L, Bertini I, Luchinat C (1991) Nuclear and electron relaxation. VCH, Weinheim

    Google Scholar 

  38. Pace CN (1986) Methods Enzymol 131:266–280

    Article  PubMed  CAS  Google Scholar 

  39. Sugio S, Kashima A, Mochizuki S, Noda M, Kobayashi K (1999) Protein Eng 12:439–446

    Article  PubMed  CAS  Google Scholar 

  40. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) J Comput Chem 25:1605–1612

    Article  PubMed  CAS  Google Scholar 

  41. La Mar GN, Yamamoto Y, Jue T, Smith KM, Pandey RK (1985) Biochemistry 24:3826–3831

    Article  PubMed  CAS  Google Scholar 

  42. Santucci R, Ascoli F, La Mar GN, Pandey RK, Smith KM (1993) Biochim Biophys Acta 1164:133–137

    PubMed  CAS  Google Scholar 

  43. Manning MC, Woody RW (1989) Biochemistry 28:8609–8613

    Article  PubMed  CAS  Google Scholar 

  44. Santucci R, Polizio F, Desideri A (1999) Biochimie 81:745–751

    Article  PubMed  CAS  Google Scholar 

  45. Ascenzi P, Bocedi A, Visca P, Altruda F, Tolosano E, Beringhelli T, Fasano M (2005) IUBMB Life 57:749–759

    Article  PubMed  CAS  Google Scholar 

  46. Myer YP, Pande A (1978) In: Dolphin D (ed) The porphyrins. Academic Press, New York, pp 271–322

  47. Goto Y, Fink AL (1994) Methods Enzymol 232:3–15

    Article  PubMed  CAS  Google Scholar 

  48. Aime S, Dastrù W, Fasano M, Arnelli A, Castagnola M, Giardina B, Ascenzi P (1992) Clin Chem 38:2401–2404

    PubMed  CAS  Google Scholar 

  49. Aime S, Fasano M, Paoletti S, Arnelli A, Ascenzi P (1995) Magn Reson Med 33:827–831

    Article  PubMed  CAS  Google Scholar 

  50. Aime S, Fasano M, Paoletti S, Cutruzzolà F, Desideri A, Bolognesi M, Rizzi M, Ascenzi P (1996) Biophys J 70:482–488

    Article  PubMed  CAS  Google Scholar 

  51. Fasano M, Baroni S, Aime S, Mattu M, Ascenzi P (2003) J Inorg Biochem 95:64–67

    Article  PubMed  CAS  Google Scholar 

  52. Muzammil S, Kumar Y, Tayyab S (1999) Eur J Biochem 266:26–32

    Article  PubMed  CAS  Google Scholar 

  53. Sattarahmady N, Moosavi-Movahedi AA, Ahmad F, Hakimelahi GH, Habibi-Rezaei M, Saboury AA, Sheibani N (2007) Biochim Biophys Acta 1770:933–942

    PubMed  CAS  Google Scholar 

Download references

Acknowledgment

The authors thank Giorgio Pariani for technical assistance. Grant MiUR FIRB RBNE03PX83 to M.C. is gratefully acknowledged.

Author information

Authors and Affiliations

  1. Dipartimento di Biologia Strutturale e Funzionale, and Centro di Neuroscienze, Università dell’Insubria, Via Alberto da Giussano 12, 21052, Busto Arsizio (VA), Italy

    Gabriella Fanali & Mauro Fasano

  2. Dipartimento di Biologia Molecolare, Cellulare ed Animale, Università di Camerino, Via Gentile da Varano III s.n.c., 62032, Camerino (MC), Italy

    Giampiero De Sanctis

  3. Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Università di Roma “Tor Vergata”, Via Montpellier 1, 00133, Rome, Italy

    Magda Gioia & Massimo Coletta

  4. Istituto Nazionale per le Malattie Infettive IRCCS “Lazzaro Spallanzani”, Via Portuense 292, 00149, Rome, Italy

    Paolo Ascenzi

  5. Laboratorio Interdisciplinare di Microscopia Elettronica, Università Roma Tre, Via della Vasca Navale 79, 00146, Rome, Italy

    Paolo Ascenzi

Authors
  1. Gabriella Fanali
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Giampiero De Sanctis
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Magda Gioia
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Massimo Coletta
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Paolo Ascenzi
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Mauro Fasano
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mauro Fasano.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Fanali, G., De Sanctis, G., Gioia, M. et al. Reversible two-step unfolding of heme–human serum albumin: a 1H-NMR relaxometric and circular dichroism study. J Biol Inorg Chem 14, 209–217 (2009). https://doi.org/10.1007/s00775-008-0439-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00775-008-0439-7

Keywords

Search

Navigation