1 H NMR relaxometric characterization of bovine lactoferrin Mauro Fasano a, * , Gabriella Fanali a , Fabio Polticelli b , Paolo Ascenzi b,c , Giovanni Antonini b,c a Dipartimento di Biologia Strutturale e Funzionale, Universit a dell’Insubria, Via Alberto da Giussano 12, I-21052 Busto Arsizio (VA), Italy b Dipartimento di Biologia, Universit a ‘‘Roma Tre’’, Viale Guglielmo Marconi 446, I-00146 Roma, Italy c Laboratorio Interdisciplinare di Microscopia Elettronica, Universit a ‘‘Roma Tre’’, Via della Vasca Navale 79, I-00146 Roma, Italy Received 9 March 2004; received in revised form 6 April 2004; accepted 7 May 2004 Available online 2 July 2004 Abstract 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  A) 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 HCO  3 . 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  A. 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  A 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 ls 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. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Lactoferrin; NMRD; Paramagnetism; Proton relaxation; Water exchange 1. Introduction Lactoferrin (Lf), also known as lactotransferrin, is a 80 kDa mammalian iron binding protein present in ex- ternal secretions, such as milk, tears, saliva, and vaginal secretions, and in polymorphonuclear leukocytes. Lf role in host defense mechanisms related to the non-immune defense system against pathogenic bacteria, fungi, and protozoa, both directly and through regulation of the inflammatory response, has been definitively established [1]. Since its first purification from milk [2–4], Lf has been recognized as an iron binding protein and identified as a member of the transferrin (Tf) family of proteins. This led to the demonstration that Lf could protect against bacterial infection through its ability to deprive bacteria of iron necessary for growth [5]. Subsequent investiga- tions have discovered other activities, some of them as- sociated with iron binding but others not. Thus, Lf is an antioxidant because its iron binding ability inhibits the iron-catalyzed formation of free radicals [6]. By virtue of its high positive charge (pI  9), Lf also has the ability to bind to various types of immune cells and microorgan- isms, and to anionic molecules such as DNA [7,8], hep- arin [9], and glycosaminoglycans [10]. Lf has two identical iron-binding sites, far from each other (44.3  A) and magnetically non-interacting. Fe(III) ions are six-coordinated, with four donor atoms pro- vided by protein sidechains (two Tyr, one His, one Asp) * Corresponding author. Tel.: +39-0331-339450; fax: +39-0331- 339459. E-mail address: mauro.fasano@uninsubria.it (M. Fasano). 0162-0134/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.jinorgbio.2004.05.003 Journal of Inorganic Biochemistry 98 (2004) 1421–1426 www.elsevier.com/locate/jinorgbio JOURNAL OF Inorganic Biochemistry