No abstract

No abstract

Human serum albumin presents in its primary structure only one free cysteine (Cys34) which constitutes the most abundant thiol of plasma. An antioxidant role can be attributed to this thiol, which is located in domain I of the protein. Herein we expressed domain I as a secretion protein using the yeast Pichia pastoris. In the initial step of ammonium sulfate precipitation, a brown pigment co-precipitated with domain I. Three chromatographic methods were evaluated, aiming to purify domain I from the pigment and other contaminants. Purification was achieved by cation exchange chromatography. The protein behaved as a noncovalent dimer. The primary sequence of domain I and the possibility of reducing Cys34 to the thiol state while avoiding the reduction of internal disulfides were confirmed by mass spectrometry. The reactivity of the thiol towards the disulfide 5,5´-dithiobis(2-nitrobenzoate) was studied and compared to that of full-length albumin. A~24-fold increase in the rate constant was observed for domain I with respect to the entire protein. These results open the door to further characterization of the Cys34 thiol and its oxidized derivatives.

No abstract

No abstract

Nitroalkene fatty acids are formed in vivo and are currently being tested in two Phase 2 clinical trials. They modulate cellular responses through Michael addition reactions with thiol‐containing proteins. Nitro‐conjugated linoleic acid (NO2‐CLA) is formed preferentially both in vivo and in vitro. There are two main positional isomers of NO2‐CLA, 9‐ and 12‐NO2‐CLA. They both react with low molecular weight thiols at two electrophilic sites located on the β‐ and δ‐carbons giving rise to reversible biphasic kinetics. The fast reaction corresponds to the formation of the β‐adduct (the kinetic product), while the slow reaction corresponds to the δ‐adduct (the thermodynamic product) (Turell, Vitturi, J. Biol. Chem., 2017). With the aim of further characterizing the biological chemistry of this bifunctional nitroalkene, we evaluated if its reaction with glutathione (GSH) is catalyzed by glutathione transferases (GST). The reaction was followed by monitoring NO2‐CLA decay at 330 nm using a stopped flow spectrophotometer. A mu‐type glutathione transferase from the parasitic platyhelminth Echinococcus granulosus (EgGST1) was used. Catalysis was observed in both phases when using a mixture of NO2‐CLA isomers. Remarkably, the δ‐addition was accelerated □13 times more than the β‐addition, 4.4 × 106 vs 3.4 × 105 M−1, respectively, at 2 mM GSH and 10 μM NO2‐CLA (25 °C, pH 7.4), suggesting specificity. To evaluate whether there is a preference of the enzyme for the NO2‐CLA isomer, purified 9‐ or 12‐ NO2‐CLA were used. No difference was observed between them. The reaction between GSH and nitro‐oleic acid (NO2‐OA), a nitroalkene fatty acid with only one electrophilic site in β, was also catalyzed by this enzyme. An acceleration of 1 × 105 M−1 was observed (2 mM GSH, 20 μM NO2‐OA), in line with the value obtained for the β‐addition of NO2‐CLA. We are now further characterizing this reaction which is expected to affect nitroalkene fatty acids metabolism and excretion and thus impact their signaling actions.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.