Protein Denaturation in Complex Solvents: Using FTIR Spectroscopy to Understand Cryopreservation

protectants are commonly used to stop ice formation in biological systems, allowing for long term preservation and storage. Dimethyl sulfoxide (DMSO) is a commonly used cryoprotectant, however; it can also be toxic to cells which limits the use of DMSO in organs. Modulating the toxicity of DMSO could aid in long term organ storage and cryopreservation, creating organ banks and dramatically reducing the size of the organ donation waitlist. Cryoprotectant toxicity neutralizers, including formamide, have been shown to aid in neutralizing the toxicity of cryoprotectants such as DMSO. However, organs are large and multifaceted which presents a host of experimental challenges. Simple proteins like lysozyme can be used to model complex organ systems. Here we use amide I infrared spectroscopy to measure thermal denaturation curves of lysozyme in ternary mixtures of DMSO, water, and formamide or dimethylformamide. We show that formamide increases the stability of lysozyme in DMSO/water mixtures, suggesting that it protects against DMSO denaturation. We also show the effects of other amides like N-methylacetamide and N-methylformamide on lysozyme. The structures of these co-solvents are shown in Figure 1. Based on their differing structures and hydrogen bonding capabilities, these amides modulate the toxicity of DMSO in different ways. This information can be used to create mixtures of co-solvents to mitigate DMSO toxicity and eventually cryopreserve biological systems.