The Antioxidant Activity of Recombinant Rat Hepatic Fatty Acid Binding Protein T94A Variant.

PURPOSE

Liver fatty acid binding protein (FABP1) is a cytoplasmic polypeptide that transports substrates throughout the cytosol and functions as an antioxidant. A common polymorphic variant, FABP1 T94A has a minor allele frequency of 26-38%, 8.3±1.9% homozygous in the human population. The purpose of this study was to mutate and isolate recombinant rat FABP1 to the T94A variant to evaluate the mutant's antioxidant activity using in vitro studies.

METHODS

Site-directed mutagenesis was used to generate a mutation in rat cDNA within a pGEX-6p-2 vector. This plasmid was transformed into competent cells and cultured for expression of FABP1 T94A mutant. The mutated protein was purified using GSTrap Fastflow columns within an ÄKTA FPLC system. A 2,7-dichlorofluorescein (DCF) assay was used to screen the T94A variant antioxidant activity. Additionally, Thiobarbituric Acid Reactive Substances (TBARS) assay was used in determining T94A mutant antioxidant activity in hydrophilic and lipophilic environments through the use of the azo compounds AAPH and MeO-AMVN, respectively and in the presence and absence of the long-chain fatty acid palmitate and α-bromo palmitate.

RESULTS

Although the FABP1 T94A (20 μM) mutant significantly reduced DCF fluorescence compared to control (no protein; P< 0.001), there were no significant difference when compared to the wild-type (WT) FABP1. T94A was able to diminish the formation of malondialdehyde (MDA) in both lipophilic and hydrophilic systems. There were significant differences between T94A mutant and WT FABP1 at concentrations 1 and 10 μM (P< 0.05) in the hydrophilic milieu, however, this was not seen at 20 μM and also not seen in the lipophilic milieu at all concentrations. When T94A was pre-incubated with the long-chain fatty acids palmitate or α -bromo palmitate, MDA formation was decreased in both lipid peroxidation systems. There were no statistical differences between the WT FABP1 and T94A bound with fatty acids in both lipid peroxidation systems, however, there was a slight statistical difference when the T94A and WT FABP1 bound α-Br-PA in the AAPH lipid peroxidation system only.

CONCLUSIONS

The T94A has antioxidant activity in both hydrophilic and lipophilic environments. The T94A variant of FABP1 does not have a loss of function in regard to acting as an antioxidant but the extent of function may be influenced by ligand binding. We conclude that populations having the minor T94A allele frequency would have similar ROS scavenging potential as those with nascent FABP1.