Biophysical Chemistry, 34 (1989) 155-162 Elsevier 155 BIOCHE 01388 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Calorimetric and circular dichroic studies of the thermal denaturation of # &lactoglobulin Savo Lapanje and NataSa Poklar Deportment of Chemistry and Chemrcol Technology. Eduord Kardel/ Unroersrty, Ljubljona, Yugoslov~o Received 1 May 1989 Revised manuscript received 3 July 1989 Accepted 3 July 1989 fl-Lactoglobulin; Protein conformation: Thermal denaturation; Calorimetry: Circular dichroism The thermal denaturatlon of &lactoglobulin in aqueous solutions at pH 5.5 and 2.0 was investigated by differential scanning calorimetry (DSC) and circular dichroic (CD) measurements. By calorimetry, the denaturation temperatures (Td), denaturation enthalpies, and specific heat capacity changes accompanying denaturation have been obtained. This allowed calculation of the enthalpy, entropy, and Gibbs free energy changes for thermal denaturation in the temperature range scanned, i.e., 20-100 ‘C. The unfolding process was found to be only partially reversible. Analysis of the far-ultraviolet CD spectra reveals that with increasing temperature the mean residue ellipticity ([19]) becomes less negative, which reflects unfolding of the native protein. At the highest temperature of CD measurements, i.e., 80 o C, conformational changes are to a large extent reversible. 1. Introduction In a previous paper [l], the denaturation of #Llactoglobulin by urea and alkylureas was re- ported. The methods used were batch calorimetry and circular dichroism (CD). The former gives rise to heat effects accompanying denaturation, while the latter reflects conformational changes. The main conclusion based on a comparison of the denaturing action of urea and alkylureas, respec- tively, was that the differences ascertained re- flected the presence of the hydrophobic moiety in the urea molecule. The subject of this investiga- tion was to examine by differential scanning calorimetry (DSC) and CD the thermal denatura- tion, i.e., the denaturing action of heat, of p- lactoglobulin in aqueous solution. The phenome- Correspondence address: S. Lapanje, Department of Chem- istry and Chemical Technology, Edvard Kardelj University, 61000 Ljubljana, Yugoslavia. non has been extensively studied by using various methods, e.g., optical rotatory dispersion [2,3], light scattering [4,5], electrophoresis [6], chromatogra- phy [7], and DSC [S-lo]. The results obtained with these methods reflected changes in various physicochemical properties brought about by heat- ing. Thus, by DSC, the denaturation enthalpy A Hd and denaturation temperature r, have been ascer- tained. The latter is the temperature of the maxi- mum heat absorption. However, for a complete thermodynamic de- scription of thermal denaturation not only AH,, and r, but also the denaturation Gibbs free en- ergy AG, and entropy AS, as well as their temper- ature dependence are needed. The thermodynamic data can be calculated from the difference in heat capacity of the denatured and native protein that can be obtained from DSC curves by a graphic . - analysis [ll-131. Since for /3-lactoglobulin data were unavailable, DSC was applied in to obtain them. these order 0301-4622/89/$03.50 0 1989 Elsevier Science Publishers B.V. (Biomedical Division)