Molecular Medicine

, Volume 14, Issue 7–8, pp 443–450 | Cite as

Purification and Characterization of Human Adrenomedullin Binding Protein-1

  • Xiaoling Qiang
  • Rongqian Wu
  • Youxin Ji
  • Mian Zhou
  • Ping Wang
Research Article


We recently discovered that the vascular responsiveness to adrenomedullin (AM), a potent vasoactive peptide, decreased during sepsis and hemorrhage in the rat and was markedly improved by its novel binding protein (AMBP-1). Moreover, AM/AMBP-1 appears to be one of the leading candidates for further development to treat sepsis and hemorrhage. However, the extremely high cost of commercial AMBP-1 limits the development of human AM and AMBP-1 as therapeutic agents. The purpose of this study was to isolate and purify AMBP-1 from normal human serum and test its stability and biological activity under in vitro and in vivo conditions. AMBP-1 was isolated and purified from normal human serum with a yield of about 3.0 mg per 100 mL and purity of >99%. The purified AMBP-1 has a AM-binding capacity similar to that of the commercial AMBP-1. Human AM and human AMBP-1 in combination significantly inhibited lipopolysaccharide-induced tumor necrosis factor (TNF)-α and interleukin (IL)-6 production from macrophages. The biological activity of the purified human AMBP-1 was well preserved when stored at 45°C for 5 d in solution or at 100°C for 1 h in powder. Moreover, administration of AM and purified AMBP-1 to hemorrhaged rats attenuated tissue injury and neutrophil accumulation. Purified AMBP-1 in combination with AM also suppressed the hemorrhage-induced rise in serum cytokines TNF-α and IL-6. Thus, we have successfully purified biologically active AMBP-1 from human normal serum and demonstrated the stability of purified human AMBP-1. This technique will enable us to further develop human AM/AMBP-1 as a novel treatment for safe and effective therapy of patients with hemorrhagic shock, sepsis, and ischemic injury.



This study was supported by National Institutes of Health grants. We thank the Proteomics Resource Center of the Rockefeller University (New York, NY) for their kind help with the mass spectrometry analysis of our samples.


  1. 1.
    Sugo S, et al. (1994) Endothelial cells actively synthesize and secrete adrenomedullin. Biochem. Biophys. Res. Commun. 201:1160–6.CrossRefGoogle Scholar
  2. 2.
    Ichiki Y, Kitamura K, Kangawa K, Kawamoto M, Matsuo H, Eto T (1994) Distribution and characterization of immunoreactive adrenomedullin in human tissue and plasma. FEBS Lett. 338:6–10.CrossRefGoogle Scholar
  3. 3.
    Kitamura K, Kangawa K, Kawamoto M, Ichiki Y, Nakamura S, Matsuo H, Eto T (1993) Adrenomedullin: a novel hypotensive peptide isolated from human pheochromocytoma. Biochem. Biophys. Res. Commun. 192:553–60.CrossRefGoogle Scholar
  4. 4.
    Lainchbury JG, Troughton RW, Lewis LK, Yandle TG, Richards AM, Nicholls MG (2000) Hemodynamic, hormonal, and renal effects of short-term adrenomedullin infusion in healthy volunteers. J. Clin. Endocrinol. Metab. 85:1016–20.PubMedGoogle Scholar
  5. 5.
    Rademaker MT, et al. (1997) Beneficial hemodynamic and renal effects of adrenomedullin in an ovine model of heart failure. Circulation 96:1983–90.CrossRefGoogle Scholar
  6. 6.
    Elsasser TH, Kahl S, Martinez A, Montuenga LM, Pio R, Cuttitta F (1999) Adrenomedullin binding protein in the plasma of multiple species: characterization by radioligand blotting. Endocrinology 140:4908–11.CrossRefGoogle Scholar
  7. 7.
    Pio R, et al. (2001) Complement factor H is a serum-binding protein for adrenomedullin, and the resulting complex modulates the bioactivities of both partners. J. Biol. Chem. 276:12292–300.CrossRefGoogle Scholar
  8. 8.
    Yang S, Zhou M, Chaudry IH, Wang P (2002) Novel approach to prevent the transition from the hyperdynamic phase to the hypodynamic phase of sepsis: role of adrenomedullin and adrenomedullin binding protein-1. Ann. Surg. 236:625–33.CrossRefGoogle Scholar
  9. 9.
    Wu R, Cui X, Dong W, Zhou M, Simms HH, Wang P (2005) Mechanisms responsible for vascular hyporesponsiveness to adrenomedullin after hemorrhage: the central role of adrenomedullin binding protein-1. Ann. Surg. 242:115–23.CrossRefGoogle Scholar
  10. 10.
    Wu R, Dong W, Zhou M, Cui X, Simms HH, Wang P (2005) Anovel approach to maintaining cardiovascular stability after hemorrhagic shock: beneficial effects of adrenomedullin and its binding protein. Surgery 137:200–8.CrossRefGoogle Scholar
  11. 11.
    Zhou M, Ba ZF, Chaudry IH, Wang P (2002) Adrenomedullin binding protein-1 modulates vascular responsiveness to adrenomedullin in late sepsis. Am. J. Physiol. Regul. Integr. Comp. Physiol. 283:R553–60.CrossRefGoogle Scholar
  12. 12.
    Zhou M, Simms HH, Wang P (2004) Adrenomedullin and adrenomedullin binding protein-1 attenuate vascular endothelial cell apoptosis in sepsis. Ann. Surg. 240:321–30.CrossRefGoogle Scholar
  13. 13.
    Sim RB, DiScipio RG (1982) Purification and structural studies on the complement-system control protein beta 1H (factor H). Biochem. J. 205:285–93.CrossRefGoogle Scholar
  14. 14.
    Wu R, Zhou M, Wang P (2003) Adrenomedullin and adrenomedullin binding protein-1 downregulate TNF-alpha in macrophage cell line and rat Kupffer cells. Regul. Pept. 112:19–26.CrossRefGoogle Scholar
  15. 15.
    Dwivedi AJ, et al. (2007) Adrenomedullin and adrenomedullin binding protein-1 prevent acute lung injury after gut ischemia-reperfusion. J. Am. Coll. Surg. 205:284–93.CrossRefGoogle Scholar
  16. 16.
    Deitch EA, Dayal SD (2006) Intensive care unit management of the trauma patient. Crit. Care Med. 34:2294–301.CrossRefGoogle Scholar
  17. 17.
    Moore FA, McKinley BA, Moore EE (2004) The next generation in shock resuscitation. Lancet 363:1988–96.CrossRefGoogle Scholar
  18. 18.
    Scalea TM, Maltz S, Yelon J, Trooskin SZ, Duncan AO, Sclafani SJ (1994) Resuscitation of multiple trauma and head injury: role of crystalloid fluids and inotropes. Crit. Care Med. 22:1610–5.CrossRefGoogle Scholar
  19. 19.
    Abou-Khalil B, Scalea TM, Trooskin SZ, Henry SM, Hitchcock R (1994) Hemodynamic responses to shock in young trauma patients: need for invasive monitoring. Crit. Care Med. 22:633–9.CrossRefGoogle Scholar
  20. 20.
    Cotton BA, Guy JS, Morris JA Jr. Abumrad NN (2006) The cellular, metabolic, and systemic consequences of aggressive fluid resuscitation strategies. Shock 26:115–21.CrossRefGoogle Scholar
  21. 21.
    Moore FA, et al. (2006) Inflammation and the host response to injury, a large-scale collaborative project: patient-oriented research core—standard operating procedures for clinical care. III. Guidelines for shock resuscitation. J. Trauma 61:82–9.CrossRefGoogle Scholar
  22. 22.
    Clifford CC (2004) Treating traumatic bleeding in a combat setting. Mil. Med. 169:8–10, 4.CrossRefGoogle Scholar
  23. 23.
    Cui X, et al. (2005) Adrenomedullin and its binding protein attenuate the proinflammatory response after hemorrhage. Crit. Care Med. 33: 391–8.CrossRefGoogle Scholar
  24. 24.
    Wu R, Wang P (2006) Preclinical studies with adrenomedullin and its binding protein as cardiovascular protective agents for hemorrhagic shock. Cardiovasc. Drug Rev. 24:204–13.CrossRefGoogle Scholar
  25. 25.
    Pelletier JP, Transue S, Snyder EL(2006) Pathogen inactivation techniques. Best Pract. Res. Clin. Haematol. 19:205–42.CrossRefGoogle Scholar
  26. 26.
    Gonzalez-Rey E, Chorny A, Varela N, Robledo G, Delgado M (2006) Urocortin and adrenomedullin prevent lethal endotoxemia by down-regulating the inflammatory response. Am. J. Pathol. 168:1921–30.CrossRefGoogle Scholar

Copyright information

© Feinstein Institute for Medical Research 2008

Authors and Affiliations

  • Xiaoling Qiang
    • 1
  • Rongqian Wu
    • 1
  • Youxin Ji
    • 1
  • Mian Zhou
    • 1
  • Ping Wang
    • 1
  1. 1.Laboratory of Surgical Research, The Feinstein Institute for Medical Research and Department of SurgeryNorth Shore University Hospital and Long Island Jewish Medical CenterManhassetUSA

Personalised recommendations