HPLC-ICP-MS for simultaneous quantification of the total and active form of the thioredoxin reductase enzyme in human serum using auranofin as an activity-based probe

Abstract

Thioredoxin reductase 1 (TrxR1) is a cytosolic and extracellular enzyme involved in cellular redox homeostasis and other important biological functions for cell survival. The presence of a selenolthiol residue in the catalytic site of the active form of TrxR1 makes it sensitive to inhibition by the Au(I) complex auranofin by means of the formation of a Se–Au complex. Therefore, in this work auranofin is proposed as an activity marker of TrxR1 through the simultaneous monitoring of Se and Au by inductively coupled plasma mass spectrometry (ICP-MS) with high performance liquid chromatography (HPLC) for species separation. The determination of the Se concentration in the TrxR1 chromatographic peak permits to obtain the total protein concentration while the Au concentration informs about the active fraction of the enzyme in biological samples such as human serum. For this purpose, TrxR1 has been isolated from the serum sample by sequential affinity (AF) and anion exchange (AE) chromatography. Then the enzyme has been incubated with auranofin under optimized conditions and analysed by AE-HPLC-ICP-MS monitoring both Au and Se. Quantification of the total enzyme concentration is carried out by post-column isotope dilution analysis (IDA) of Se, while quantification of the active form of the TrxR1 (labelled with auranofin) is conducted by using a gold-containing generic standard. By taking into account the stoichiometry for the active enzyme : auranofin (1 : 4) complex it is possible to determine the fraction of the active protein in the sample. The developed methodology allows a novel simultaneous determination of both, the total enzyme concentration and the active concentration of TrxR1 at very low levels in human serum. The results obtained seem to indicate that only a small amount (about 9%) of the enzyme present in serum is in its active form.