Thermal Stabilization of Proteins by Mono- and Oligosaccharides: Measurement and Analysis in the Context of an Excluded Volume Model Ilyas Beg, † Allen P. Minton,* ,‡ Md. Imtaiyaz Hassan, † Asimul Islam, † and Faizan Ahmad* ,† † Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, Jamia Nagar, New Delhi 110025, India ‡ National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, United States * S Supporting Information ABSTRACT: The reversible thermal denaturation of apo α- lactalbumin and lysozyme was monitored via measurement of changes in absorbance and ellipticity in the presence of varying concentrations of seven mono- and oligosaccharides: glucose, galactose, fructose, sucrose, trehalose, raffinose, and stachyose. The temperature dependence of the unfolding curves was quantitatively accounted for by a two-state model, according to which the free energy of unfolding is increased by an amount that is independent of temperature and depends linearly upon the concentration of added saccharide. The increment of added unfolding free energy per mole of added saccharide was found to depend approximately linearly upon the extent of oligomerization of the saccharide. The relative strength of stabilization of different saccharide oligomers could be accounted for by a simplified statistical-thermodynamic model attributing the stabilization effect to volume exclusion deriving from steric repulsion between protein and saccharide molecules. O ver the course of its lifetime, an organism may experience significant variation in environmental parameters such as temperature, pressure, salinity, and pH. A change in any of these variables is known to affect the functional properties of biological macromolecules and could in principle interfere with or inhibit essential processes necessary for the sustenance of life. 1-4 The process of evolution has therefore resulted in a variety of strategies for the protection of macromolecules from deleterious effects arising from environmental stress. One of these strategies is the accumulation of small organic molecules termed osmolytes 5-9 within cells and in extracellular fluids. In vitro experiments have shown that these molecules provide increased stability to proteins and other cell components against various stress conditions and maintain the normal functioning of the organism. 6,10,11 Chemically, osmolytes are classified into (i) amino acids and their derivatives, (ii) methylamines, and (iii) sugars and polyols. 6,12 The mechanism underlying the stabilization of proteins by osmolytes has been the subject of extensive study. 10,13-25 On the basis of measurements of the free energy of transferring amino acid side chains and the peptide backbone from water to an osmolyte solution, Bolen and co-workers proposed a generalized physicochemical mechanism for the stabilization of proteins by osmolytes termed the “osmophobic effect”. 19-24 This effect derives from a highly unfavorable interaction of osmolytes with peptide backbone. Because unfolding of the protein results in an increased level of exposure of the backbone to solvent, the free energy of the denatured state is increased relative to that of the native state, shifting the equilibrium between native and denatured states toward the native state. In 1981, Lee and Timasheff 26 reported the first quantitative study of the thermal stabilization of three proteins, α- chymotrypsin, chymotrypsinogen, and ribonuclease, by a saccharide, sucrose. Each of the proteins appeared to behave in accordance with a model in which the protein exists as a mixture of two thermodynamic states, native and unfolded, in dynamic equilibrium. The temperature at which each protein was observed to be half-folded, i.e., at which the equilibrium constant for unfolding was equal to unity, was observed to increase linearly with sucrose concentration. A van’t Hoff analysis of the temperature dependence of the equilibrium constant for unfolding at different sucrose concentrations indicated that sucrose did not significantly alter the enthalpy change accompanying unfolding of any of the three proteins; i.e., within experimental precision, the change in stability was due entirely to a decrease in the entropy change associated with unfolding. Careful measurement of the dependence of the partial specific volume of protein upon sucrose concentration Received: April 17, 2015 Revised: May 21, 2015 Article pubs.acs.org/biochemistry © XXXX American Chemical Society A DOI: 10.1021/acs.biochem.5b00415 Biochemistry XXXX, XXX, XXX-XXX