Fig 7. Reduced Trx2 and 5S-Trx2 prevent thermal aggregation of luciferase.
(A) Native luciferase (0.1 μM) was incubated in the absence or presence of different molar ratios of either Trx2red, Trx2ox or 5S-Trx2 at 44°C. Thermal aggregation of luciferase was followed by measuring light scattering at 360 nm. Trx2red and Trx2ox were prepared as described in Material and methods. For measuring the effect of Trx2red, the assay buffer was supplemented with 0.2 mM DTT which did not affect aggregation of luciferase in the absence of Trx2 or presence of 5S-Trx2. (B) Thermal aggregation of luciferase in the absence of Trx2 was set as 100% and the percentage of light scattering in the presence of a 5:1, 10:1 or 20:1 molar ratio of the different Trx2 species to luciferase was calculated from at least three independent assays. Student’s t-test (one-sample, unpaired) was used to evaluate significant differences between Trx2 and 5S-Trx2 (* = p < 0.05). (C) Trx2red (20 μM) was pre-incubated in assay buffer at 44°C in the presence or absence of 2 mM DTT. After different times, the volume was increased ten-fold to the final assay volume, luciferase was added (0.1 μM), and light scattering was measured.