The temperature dependence of the heat capacity function of holo and apo alpha-lactalbumin has been studied by high sensitivity differential scanning microcalorimetry. The heat capacities of the holo and apo forms in the native state were found to be close to, but somewhat higher than, that of lysozyme, which has a similar structure. At pH values higher than 5, the heat-denatured state and the unfolded state are indistinguishable. At lower pH values, the heat capacity of the state obtained by heat or acid denaturation is lower than what is expected for the completely unfolded polypeptide chain, but it approaches that value at higher temperatures. The heat capacity increment of the denatured state correlates well with the amount of residual structure measured by ellipticity (i.e., the lower the residual structure, the higher the heat capacity). The extent of residual structure in the denatured state, which is exceptionally high in alpha-lactalbumin, decreases upon increasing temperature and at approximately 110 degrees C becomes close to that observed in 6 M GdmCl. Above 110 degrees C, the denatured state of alpha-lactalbumin is practically indistinguishable in heat capacity and ellipticity from the fully unfolded state. The calorimetric data have been analyzed quantitatively using a statistically thermodynamic formalism. This analysis indicates that the long-range or global cooperativity of the protein is lost after heat denaturation of the native state, causing the remaining elements of residual structure to behave in a more or less independent fashion. At pH values close to neutral, heat denaturation occurs at high temperature and yields a totally unfolded polypeptide with no measurable population of partly folded intermediates. At lower pH values, denaturation occurs at lower temperatures and a progressively higher population of intermediates is observed. At pH 4.2, about 50% of the molecules is in compact intermediate states immediately after heat denaturation; however, at pH 3.5, this percentage is close to 80% and at pH 3.0 it reaches about 100% of the protein molecules. Upon heating, the unfolded state progressively becomes the predominant species. The analysis of the heat capacity data for alpha-lactalbumin indicates that the best model to account for the observed behavior is one in which the denatured state is represented as a distribution of substates with varying degrees of residual structure. At low temperatures, the distribution is centered around rather compact substates with significant residual structure. At higher temperatures, the distribution shifts toward states with less residual structure and eventually to the completely unfolded state.

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The thermodynamics of barnase denaturation has been studied calorimetrically over a broad range of temperature and pH. It is shown that in acidic solutions the heat denaturation of barnase is well approximated by a 2-state transition. The heat denaturation of barnase proceeds with a significant increase of heat capacity, which determines the temperature dependencies of the enthalpy and entropy of its denaturation. The partial specific heat capacity of denatured barnase is very close to that expected for the completely unfolded protein. The specific denaturation enthalpy value extrapolated to 130 "C is also close to the value expected for the full unfolding. Therefore, the calorimetrically determined thermodynamic characteristics of barnase denaturation can be considered as characteristics of its complete unfolding and can be correlated with structural features -the number of hydrogen bonds, extent of van der Waals contacts, and the surface areas of polar and nonpolar groups. Using this information and thermodynamic information on transfer of protein groups into water, the contribution of various factors to the stabilization of the native structure of barnase has been estimated. The main contributors to the stabilization of the native state of barnase appear to be intramolecular hydrogen bonds. The contributions of van der Waals interactions between nonpolar groups and those of hydration effects of these groups are not as large if considered separately, but the combination of these 2 factors, known as hydrophobic interactions, is of the same order of magnitude as the contribution of hydrogen bonding.Keywords: barnase; hydrogen bonding; hydrophobic interactions; scanning microcalorimetry Barnase attracts the special interest of researchers engaged in studying protein folding because it is one of the smallest globular proteins (1 10 amino acid residues; see Kinemage 1) that does not have disulfide crosslinks but is quite stable and unfolds reversibly. The latter circumstance is very important for quantitative analysis of this process by equilibrium thermodynamics, specifying the stability of the native state in energetic terms.In solutions close to neutral pH, reversible unfolding of barnase by denaturants and temperature appears to be a 2-state transition (Hartley, 1968(Hartley, , 1975Makarov et al., 1993). Our earlier unpublished calorimetric studies of the heat denaturation of barnase, carried out at the Protein Research Institute in Russia in 1989, showed also that this process is approximated well by a 2-state transition. However, later in 1993 the paper of Makarov et al. (1993) ing subdomains. Analysis of the 3-dimensional structure of these proteins does indeed reveal 2 hydrophobic clusters (see Kinemage 1;Serrano et al., 1992). Therefore, the mode of barnase denaturation required detailed investigation. Because there was also some deviation between our estimates of the enthalpy of denaturation and that of Makarov et al. (1993), we felt it necessary to repeat calorimetric measurements on barnase to ...