Purification of human thioredoxin reductase: Properties and characterization by absorption and circular dichroism spectroscopy

Abstract

The flavoenzyme thioredoxin reductase (TR) and its natural substrate thioredoxin comprise a redox system generally found in all organisms. In order to better understand the biochemistry of this redox system, TR was purified (> 4000-fold) from human placenta as a dimer of 60-kDa subunits. The molecular size of native TR was determined to be 160 kDa by gel filtration chromatography whereas migration on a sucrose gradient gave a molecular mass of 130 kDa. The pI of TR was determined to be 4.85. The temperature optima for DTNB and insulin reduction by TR were 52 and 40 degrees C, respectively. Preincubation of TR at 60 degrees C for up to 1 h showed no decrease in the enzymatic rates when assayed at 28 degrees C, while temperatures above 65 degrees C resulted in an irreversible loss of activity. Circular dichroism (CD) spectra of TR indicated that the secondary structural changes at 60 degrees C were only partly reversible at 28 degrees C. CD studies showed the flavoenzyme had a TM of 63 degrees C and above 45 degrees C began to exhibit changes in the secondary structure. Equilibrium denaturation of TR by temperature and guanidine hydrochloride suggested that FAD was not displaced during inactivation of TR and that the tertiary structure was primarily disrupted prior to denaturation of the secondary structure. The results of this study show that purified human TR is a relatively thermostable flavoenzyme whose tightly bound FAD group is not displaced by elevated temperatures up to 60 degrees C or by relatively low concentrations of guanidine hydrochloride.

A comment on the absence of calcium regulation of human thioredoxin reductase

“…We have recently reported the purification and characterization of human TR from placental tissue [12]. In contrast to the earlier reports, we have now determined that human TR activity is not affected by Ca 2+ up to mM levels.…”

Identification and Functional Characterization of Thioredoxin from Trypanosoma brucei brucei

“…T. brucei thioredoxin is readily reduced by human thioredoxin reductase. The K m value of 6 M derived from a Lineweaver-Burk plot (not shown) compares well with those for human and E. coli thioredoxin, which are 4.3 and 20 M, respectively (43). To test if trypanothione reductase is able to reduce thioredoxin, we replaced human thioredoxin reductase by T. cruzi trypanothione reductase in the NADPH/thioredoxin/insulin assay (data not shown).…”

Biochemical, structural, and biological properties of human thioredoxin active site peptides

“…Recombinant human Trx and human placental TR were prepared as previously described [17,20]. Two assays were performed to monitor TR activity.…”