Calorimetric and Raman investigation of cow’s milk lactoferrin Michele Iafisco • Ismaela Foltran • Michele Di Foggia • Sergio Bonora • Norberto Roveri AICAT2010 Special Chapter Ó Akade ´miai Kiado ´, Budapest, Hungary 2010 Abstract Lactoferrin (LF), a non-heme iron-binding protein of blood plasma and milk with antioxidant, cario- static, anticarcinogenic, and anti-inflammatory properties, has been studied by differential scanning calorimetry (DSC) and Raman spectroscopy over a wide pH range (4.0–9.0). Using these two techniques, the modifications in the quantity of iron bounded in the cow’s milk LF and in the secondary structures, as a function of pH and heating, have been evaluated. DSC curves showed higher value of denaturation temperatures and enthalpy changes when LF was saturated with iron (holo-form) than when it was in its unsaturated form (apo-form). The denaturation curves of the protein solutions at pH C 5.5 confirming that LF is a mix of apo- and holo-forms; on the contrary at pH 4.0, the holo-form is practically absent. Spectroscopic investigation showed that, as a function of pH, the content of a-helix increases up to pH 7.4, followed by a small decrease by further pH increase. The b-sheet percentage exhibits the opposite behavior, while the random-coil and turn struc- tures do not change noticeably. In contrast, after heat- induced denaturation, strong variations were observed in the secondary structure, with an evident increase of b-sheet and decrease of the a-helix percentage. Finally, both ther- mal and spectroscopic analysis pointed out that the struc- ture of cow’s milk LF is strictly sensible to pH variation and it has the highest thermal stability at physiological pH. Keywords Lactoferrin  Raman spectroscopy  Differential scanning calorimetry  Protein denaturation  Protein biophysics Introduction Lactoferrin (formerly known as lactotransferrin) (LF) is a non-heme iron-binding protein that is a part of the trans- ferrin protein family, thus belonging to those protein capable of binding and transferring Fe 3? ions in blood serum [1]. LF is a glycoprotein with a molecular weight of about 80 kDa and has a basic isoelectric point (IP) (*9.0) [2]. It consists of a single polypeptide chain containing 703 amino acids folded into two symmetrical lobes (N and C lobes), which are highly homologous with each other (33–41% homology). These two lobes are connected by a hinge region containing parts of a a-helix between amino acids 333 and 343 in human LF. The polypeptide chain includes amino acids 1–332 for the N-lobe and 344–703 for the C-lobe, and the protein structure creates two domains for each lobe [3]. There are three forms of LF according to its iron satu- ration: apolactoferrin (iron free), monoferric form (one ferric ion), and hololactoferrin (binds two Fe 3? ions); the M. Iafisco (&)  I. Foltran  N. Roveri Dipartimento di Chimica ‘‘G. Ciamician’’, Alma Mater Studiorum, Universita ` di Bologna, Via Selmi 2, 40126 Bologna, Italy e-mail: michele.iafisco@unibo.it I. Foltran e-mail: ismaela.foltran@unibo.it M. Iafisco  I. Foltran Dipartimento di Scienze Mediche, Universita ` del Piemonte Orientale, Via Solaroli 4, 28100 Novara, Italy M. Di Foggia  S. Bonora Dipartimento di Biochimica ‘G. Moruzzi’ Alma Mater Studorium, Universita ` di Bologna, Via Belmeloro 8/2, 40126 Bologna, Italy 123 J Therm Anal Calorim (2011) 103:41–47 DOI 10.1007/s10973-010-1084-2