Michigan Technological University
Department of Chemistry
John Hausman
Advisor: Dr. Ashutosh Tiwari
Master’s Defense
Monday August 10, 2015
9:30 am, Chem-Sci 101
ON THE PROTECTIVE PROPERTIES OF GLYCINE BASED OSMOLYTES IN A THIOL REDUCING ENVIRONMENT
The protective properties of osmolytes have been studied intently for decades. Originally used to aid in the crystallization of proteins in x-ray diffraction studies, these cosolvents have been shown to reverse protein denaturation and aggregation. Osmolytes aid extremophiles in surviving harsh environments by preferentially excluding themselves from the surface of the protein, thus directing water molecules to the protein’s surface. Their use is increasing and affects the everyday consumer as these osmolytes are naturally found in many health foods and also in many daily use products such as shampoo. Consumers also supplement their diets with thiol-based antioxidants such as glutathione as part of healthier life style. However, thiol-based antioxidants in high concentration have been known to cause un-intended health issues that relate to its disulfide reducing property. The disulfide-reducing agent can cleave the proteins’ disulfide-bonds and promote misfolding and aggregation. In this thesis, we investigated the influence of commonly consumed glycine-based osmolytes on providing stability to proteins against a disulfide-reducing agent. We chose glycine, sarcosine (N-methyl glycine), di-methyl glycine (DMG), and betaine (N,N,N-trimethylglycine) with increasing number of methyl groups. An added benefit to studying this class of osmolytes is to also investigate the impact N-methyl substitution has on the osmolytes’ protective properties. We studied the effect of these osmolytes on protein aggregation using spectroscopic techniques such as UV-visible absorbance, intrinsic fluorescence and extrinsic fluorescence measurements. In addition, we carried out non-reducing SDS-PAGE to check for higher order aggregates and characterized morphology of these aggregates using scanning electron microscopy. Overall, our results show that of all the osmolytes used, glycine was the best stabilizer followed by sarcosine. Betaine and dimethylglycine did not provide effective protection against disulfide-reducing influence.