טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentAvi Shpigelman
SubjectMechanisms of Saccharide Effect on PNIPA Behavior in
Aqueous Media as a Model for Water-Saccharide-
Protein Systems
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Professor Livney Yoav
Full Thesis textFull thesis text - English Version


Abstract

The stability of proteins in aqueous solutions is of great importance in biotechnology & food science. Saccharides as co-solutes tend to provide protection to proteins against denaturation induced by heat, freezing or drying, but the mechanisms of these protective effects are still not fully understood. With the aim of shedding some new light on this intriguing problem, we studied the effects of saccharide structure in aqueous solutions on poly-N-isopropyl acrylamide (PNIPA), as a simple model representing certain key behaviours of proteins. Using a number of systematically selected sugars, we aimed at relating structural characteristics of these co-solutes to their effect on PNIPA solution behaviour. Using Isothermal Titration Calorimetry (ITC) we showed that the main effect of saccharides on the polymer can not be attributed to direct binding of the sugars to the polymer. Using Differential Scanning Calorimetry (DSC) we studied the slopes (“K”) of decreasing polymer coil-globule-transition (CGT) temperature (“cloud point”) with rising sugar concentration. Beyond the relatively expected observation that steric exclusion is important, we observed previously unreported significant differences between the effects of saccharide isomeres.  Additionally we studied binary water sugar systems to determine the hydrated volume, isentropic compressibility and hydration numbers. Our results greatly support our hypothesis that binary sugar solution properties can be good describers of the sugar effect on PNIPA phase transition in the ternary solution. We suggest that stereochemically, galactose has a worse fit to water structure, and it binds water molecules around it more compactly than mannose and glucose. We found a good correlation between the effect of the isomer on the CGT of PNIPA, the partial molar isentropic compressibility and the hydration number of this sugar. We conclude that the larger (in terms of number of water molecules) and the more compact the hydrated cluster a sugar forms, the worse a co-solvent it is for the polymer, and the stronger its lowering effect of the CGT. Such favoring of the compact globular state of the polymer by the sugar provides a protective effect against heat denaturation for globular proteins. We used a previously described FTIR method to study the effect of glucose on the functional groups of the polymer. FTIR is evidently a good method for studying sugar effect on the CTG of PNIPA. While heating the solution we observed indications that glucose induced earlier dehydration of both the hydrophilic and hydrophobic groups.