Lysozyme Denaturation in DMSO Mixtures
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Lysozyme is a small, well characterized protein commonly used to study denaturation. Proteins like lysozyme can be used as model systems to simplify complicated phenomena such as cyropreservation- the formation of a glass transition state in a process known as vitrification. Cryoprotectants (CPAs) prevent ice formation and subsequent cell death by forming hydrogen bonds with water. Dimethyl sulfoxide (DMSO) is one such cryroprotectant commonly used in many laboratories, but it is known to be toxic at high concentrations. A new area of interest involves solvents called cryoprotectant toxicity neutralizers (CTNs) which have been hypothesized to interact with DMSO and reduce its toxic effects on living systems. Formamide is one such CTN and its interactions with DMSO and proteins have not been thoroughly studied. The ability to vitrify organs and extend their brief ex-vivo lifetimes would have dramatic impacts, but the long term preservation of tissues at cryogenic tempeartures has been hindered by issues with toxicity. In this research, we performed temperature dependent infrared absorption spectroscopy of the Amide I backbone of lysozyme (which serves as a spectrscopic prove to reveal its secondary structure) to elucidate the interactions of CTNs and CPAs. Spectra were taken from 5 to 90 C, and the melting temperature of lysozyme in different mixtures of DMSO, formamide, and water were calculated using singular value decomposition (SVD) analysis. The thermal denaturation curves of lysozyme were fit to sigmoidal functions, and a clear two-state transition was observed. Increasing DMSO concentration reduced the melting point of lysozyme, as it grew less stable and DMSO acted as a denaturant. Adding small volumes of formamide led to an increase in melting temperature, which showed that formamide could be acting as a CTN and stablizing the system. These results signify the first step towards understanding the effects of CTNs and CPAs on biological systems and are vital to moving the field forward on a molecular level. Further studies using fluorescence and calorimetry can be done to reveal the nature of these interactions.