Contents lists available at ScienceDirect Biophysical Chemistry journal homepage: www.elsevier.com/locate/biophyschem Comparison of the thermal stabilization of proteins by oligosaccharides and monosaccharide mixtures: Measurement and analysis in the context of excluded volume theory Ilyas Beg a , Allen P. Minton b, ⁎ , Asimul Islam a , Md Imtaiyaz Hassan a , Faizan Ahmad a, ⁎⁎ a Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, Jamia Nagar, New Delhi 110025, India b Laboratory of Biochemistry and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA HIGHLIGHTS • Denaturation of proteins was studied in the presence of monosaccharides mixtures. • Monosaccharides have more stabi- lizing effect than their respective oli- gosaccharides. • Stabilizing effect of sugar mixtures is accounted by excluded volume model. GRAPHICAL ABSTRACT ARTICLE INFO Keywords: Excluded volume effect Osmolytes Monosaccharides Oligosaccharides Degree of stabilization ABSTRACT The thermal stability of apo α–lactalbumin (α–LA) and lysozyme was measured in the presence of mixtures of glucose, fructose, and galactose. Mixtures of these monosaccharides in the appropriate stoichiometric ratio were found to have a greater stabilizing effect on each of the two proteins than equal weight/volume concentrations of di- tri- and tetra- saccharides with identical subunit composition (sucrose, trehalose, raffinose, and stachyose). The excluded volume model for the effect of a single saccharide on the stability of a protein previously proposed by Beg et al. [Biochemistry 54 (2015) 3594] was extended to treat the case of saccharide mixtures. The extended model predicts quantitatively the stabilizing effect of all monosaccharide mixtures on α–LA and lysozyme reported here, as well as previously published results obtained for ribonuclease A [Biophys. Chem. 138 (2008) 120] to within experimental uncertainty. 1. Introduction With the exception of urea, all naturally occurring osmolytes sta- bilize proteins with respect to unfolding under denaturing stress conditions [1]. Stabilizing osmolytes interact unfavorably with both native (N) and unfolded (U) states of the thermodynamic equilibrium (N ↔ U), but more unfavorably with the U state, thus shifting the equilibrium between the two states toward the N state [2,3]. The effect https://doi.org/10.1016/j.bpc.2018.03.002 Received 9 March 2018; Received in revised form 16 March 2018; Accepted 16 March 2018 ⁎ Correspondence to: A. Minton, C Laboratory of Biochemistry and Genetics, National Institute of Diabetes and, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA. ⁎⁎ Correspondence to: F. Ahmad, Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi 110025, India. E-mail addresses: minton@helix.nih.gov (A.P. Minton), fahmad@jmi.ac.in (F. Ahmad). Abbreviations: Apo, α–lactalbumin; α–LA, Ribonuclease A; RNase, Guanidinium chloride; GdmCl, Glucose; Glc, Galactose; Gal, Fructose, Fru Biophysical Chemistry 237 (2018) 31–37 Available online 22 March 2018 0301-4622/ © 2018 Elsevier B.V. All rights reserved. T