Thermodynamic stability of porcine b-lactoglobulin A structural relevance Tatiana V. Burova 1 , Natalia V. Grinberg 1 , Ronald W. Visschers 2,3 , Valerij Y. Grinberg 1 and Cornelus G. de Kruif 2,4 1 Institute of Biochemical Physics, Russian Academy of Sciences, Moscow, Russia; 2 NIZO food research, Ede, the Netherlands; 3 Wageningen Centre for Food Science, the Netherlands and 4 Van’t Hoff Laboratory, Debye Institute, Utrecht University, the Netherlands The proposed biological function of b-lactoglobulins as transporting proteins assumes a binding ability for ligands andhighstabilityundertheacidicconditionsofthestomach. This work shows that the conformational stability of non- ruminant porcine b-lactoglobulin (BLG) is not consistent withthishypothesis.ThermaldenaturationofporcineBLG was studied by high-sensitivity differential scanning calori- metry within the pH range 2.0–10.0. Dependences of the denaturation temperature and enthalpy on pH were obtained, which reveal a substantial decrease in both parameters in acidic and basic media. The denaturation enthalpy follows a linear dependence on the denaturation temperature. The slope of this line is 9.4 ± 0.6 kJÆmol )1 Æ K )1 , which is close to the denaturation heat capacity increment D d C p ¼ 9.6±0.5kJÆmol )1 ÆK )1 , determined directlyfromthethermograms.AtpH 6.25thedenaturation temperatures of porcine and bovine BLG coincide, at 83.2 °C. At this pH the denaturation enthalpy of porcine BLGis300kJÆ mol )1 .Thedenaturationtransitionofporcine BLGwasshowntobereversibleatpH3.0andpH9.0.The transition profile at both pH values follows the two-state modelofdenaturation.BasedonthepH-dependenceofthe transition temperature and the linear temperature depend- ence of the transition enthalpy, the excess free energy of denaturation, D d G E , of porcine BLG was calculated as a function of pH and compared with that of bovine BLG derivedfrompreviouslyreporteddata.ThepH-dependence of D d G E isanalysedintermsofthecontributionsofside-chain H-bonds to the protein stability. Interactions stabilizing nativefoldsofporcineandbovineBLGarediscussed. Keywords: b-lactoglobulin; porcine; stability; thermody- namics;DSC. The protein b-lactoglobulin (BLG) has a long story of comprehensivestudiesofitsphysicochemicalandbiological aspectsbutitstillremainsaproteinwithundefinedfunction. The most widespread hypothesis of its biological function refers to the role of BLG as a transport protein [1,2]. This view is supported by numerous data on binding of hydro- phobicligandstobovineBLG[3–6].Structuraldataencour- age this idea, providing an indication of possible binding sites for retinol and fatty acids in bovine BLG [1,6–8]. Bovine BLG stands out because of its high structural and proteolyticstabilityatlowpH[1,6–16]butitreadilylosesits quaternaryandtertiarystructureatweaklybasicpH[17–22]. These features are believed to play a protecting role for bound ligands under acidic conditions in the stomach and afford their release in the basic intestine [1]. In the light of theseconcepts,informationontheconformationalstability of BLG at different pH becomes of interest for functional considerations. Particularly, more light could be shed on this problem by involving nonruminant b-lactoglobulins that are known to differ from bovine BLG in their quaternary structure and binding properties [4,5,23–25]. Porcine BLG shows 66% sequence identity with bovine BLG[26].Itcontainstwodisulfidebondsbutdoesnothave the free thiol group at Cys121. The latter property attracts anadditionalinteresttoporcineBLGasanaturallyexisting model for studying the role of the free thiol in postdena- turation processes of bovine BLG and, finally, in milk processing [27–29]. It is well known that thermal denatur- ation of bovine BLG at neutral and weakly basic pH is a complex,irreversibleprocess.Itinvolvesthedissociationof bovine BLG dimer [20,30], unfolding and aggregation [21,31–35]. One of the most important consequences of these conformational changes is the exposure of the free thiol(Cys121)ofbovineBLG,whichishighlyreactiveand initiates the formation of intermolecular disulfide bonds [19,36–38]. Disulfide-mediated aggregation is considered to be the key factor of thermotropic gelation of bovine BLG. Inaccordancewiththispoint,porcineBLGwasreportedto beunabletoformthermallyinducedgels[39]. Porcine BLG remains up to now one of the poorly studied b-lactoglobulins. This is a small globular protein withamolecularmassof18.5kDaandpIof4.6[23,40,41]. Itsaminoacidsequencecomposedof160residueshasbeen determined [42] and two main genetic variants designed as porcine BLG A and C were identified [40]. Recently the three-dimensional structure of this protein was reported [26]. Porcine BLG reveals a pH-dependent dimerization Correspondence to C.G.deKruif,NIZOfoodresearch, Kernhemseweg2,POBox20,6710BAEde,theNetherlands. Fax: + 31 318650 400; E-mail: dekruif@nizo.nl Abbreviations:BLG, b-lactoglobulin;HS-DSC,high-sensitivity differentialscanningcalorimetry (Received29April2002,revised23June2002,accepted28June2002) Eur. J. Biochem. 269, 3958–3968 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03081.x