1H NMR relaxometric characterization of bovine lactoferrin

Mauro Fasano, Gabriella Fanali, Fabio Polticelli, Paolo Ascenzi, Giovanni Antonini

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Lactoferrin (Lf) is a mammalian iron binding protein present in external secretions and in polymorphonuclear leukocytes. Its role in host defense mechanisms related to the non-immune defense system has been definitively established. Lf has two identical iron-binding sites, far from each other (44.3 Å) and magnetically non-interacting. Fe(III) ions are six-coordinated, with four donor atoms provided by protein sidechains (two Tyr, one His, one Asp) and two oxygen atoms from a bridged HCO3 -. This set of ligands provides an ideal coordination scheme for stable and reversible iron binding. Nuclear magnetic relaxation dispersion (NMRD) profiles of Lf are consistent with a closest distance for a single water hydrogen atom of 3.1 Å. By looking at the X-ray structure of Lf (PDB ID code: 1BLF) we can locate two water oxygens at 3.95 and 4.27 Å from each Fe(III), respectively. Temperature dependence data suggest that an important contribution to the overall paramagnetic contribution to the solvent water relaxation rate arises from one or more second sphere water molecules in slow exchange with the bulk. A decreasing value of the exchange rate is obtained, ranging from 1.2 to 0.7 μs in the observed temperature range (25-65°C), with an activation enthalpy of 7.3±0.8 kJ mol-1. The low exchange rate obtained from NMRD data can be explained by the observation that both water molecules are bound to several polar groups of the protein backbone and side chains. By increasing the pH from 6.5 to 12 two distinct titrations are observed, consistent with sequential removal of both water molecules.

Original languageEnglish
Pages (from-to)1421-1426
Number of pages6
JournalJournal of Inorganic Biochemistry
Issue number8
Publication statusPublished - Aug 2004


  • Lactoferrin
  • NMRD
  • Paramagnetism
  • Proton relaxation
  • Water exchange

ASJC Scopus subject areas

  • Biochemistry
  • Inorganic Chemistry


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