Application and Mechanism of Malonic Acid as a Green Alternative for Protein-Crosslinking

Formaldehyde fixation is the main method for crosslinking cellular proteins prior to their usage in immunocytochemistry. In order to create these links, formaldehyde undergoes a Mannich reaction in which the formaldehyde forms a methylene bridge between the aminogroup of two amino acids. Crosslinking increases protein stability allowing for more accurate preservation of in vivo conformations which in turn increases binding affinity of fluorochrome conjugated antibodies for fluorescent imaging. Formaldehyde is also a known carcinogen as classified by the National Cancer Institute. Malonic acid, a green, plant-based, water-soluble, and relatively inexpensive polycarboxylic acid has been shown to undergo crosslinking of proteins through an unknown mechanism. To test whether malonic acid can crosslink proteins within cells we fixed SH-5YSY cells with either malonic acid or formaldehyde and then stained with a fluorochrome conjugated antibody for the cytoskeletal protein α-tubulin. The cells were then imaged 72 hours after fixation. We observed a non-significant difference in the fluorescence of immunostained SH-5YSY cells fixed with malonic acid as compared to paraformaldehyde (p-value = 0.2469, ANOVA). In addition, we have created a theoretical mechanism showing malonic acid forming a propyl bridge for crosslinking proteins in a similar mechanism to that of formaldehyde. Here, we show that malonic acid is able to fix cells and retain fluorescence just as well as paraformaldehyde up to 72 hours after fixation and present several possible mechanisms for this chemical process.