TITLE:
Application and Mechanism of Malonic Acid as a Green Alternative for Protein-Crosslinking
AUTHORS:
Donald Szlosek, Douglas Currie
KEYWORDS:
Green Chemistry, Protein Fixation, Protein Crosslinking, Cellular Fixation, Formaldehyde Fixation, Fixation, Malonic Acid
JOURNAL NAME:
Green and Sustainable Chemistry,
Vol.6 No.2,
May
24,
2016
ABSTRACT: 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.