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Raman spectroscopy can be a powerful tool for the characterization of modified amino acids and proteins. In addition to the potential for quantitative results, it offers the advantage of not requiring any sample preparation. Modification of amines and thiols on amino acids and proteins are common reactions used for medical, biological, food, and agricultural purposes. We hypothesized that the Raman spectrum could be used to quantify the reactions and would be more informative than typical characterization techniques such as the ninhydrin test. To prove the hypothesis, the amino acids alanine, cysteine, and lysine were modified with ethyl vinyl sulfone (EVS) using a nucleophilic addition reaction known as the Michael addition and the product was characterized using Raman spectroscopy. The Raman spectroscopy results were compared with ultraviolet-visible spectroscopy results based on ninhydrin analysis of the modified amino acids. The Raman spectroscopy analysis was able to discern site-specific reactions on the amino acids and suggested that more amino acid moieties were substituted than predicted using the ninhydrin test alone. Substitution of the full protein ovalbumin with EVS showed similar results. The ninhydrin test showed the substitution of primary amines and thiols but could not detect substitution of secondary amines remaining after loss of the primary amine.

When proteins are removed from their native state they suffer from two deficiencies: (1) glassy behavior with glass transition temperatures (Tg) well above room temperature and (2) thermal instability. The glassy behavior originates in multiple hydrogen bonds between amino acids on adjacent protein molecules. Proteins, like most biopolymers, are thermally unstable. Substituting ovalbumin with linear and cyclic substituents using a facile nucleophilic addition reaction can affect Tg and thermal stability. More hydrophobic linear substituents lowered Tg by interrupting intermolecular interactions and increasing free volume. More hydrophilic and cyclic substituents increased thermal stability by increasing intermolecular interactions. In some cases, substituents instituted cross-linking between protein chains that enhanced thermal stability. Internal plasticization using covalent substitution and external plasticization using low molecular weight polar liquids show the same protein structural changes and a signature of plasticization is identified.

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