Figure 3.

A2 can be used to quantify TTR subunit exchange in blood plasma. (A) Samples were incubated with A2 or vehicle control for 3 h at 25 °C. The recombinant WT TTR homotetramer peak in standard phosphate buffer (···) is detected at an elution time of 4.5 min. In healthy donor plasma (green line), a similar peak is detected at 4.25 min. This TTR·A2 conjugate peak is absent in plasma from a TTR knockout (KO) mouse (orange line) and in human plasma that was incubated with vehicle without A2 (purple line). In all plasma samples, A2 also detects a peak eluting around 1–3 min (peak 0). The inset shows the structure of A2. (B) Healthy donor plasma was incubated at 25 °C without (red) or with 10 μM tafamidis (black). At time zero, A2 (30 μM) was added, and the sample was diluted with 5 volumes of sodium phosphate buffer (50 μM, pH 7.6) and immediately placed in the UPLC autosampler to acquire the first time point. Subsequent time points were acquired automatically from the same vial at the indicated times. The area of peak 1 was calculated for each time point. Symbols represent means; error bars represent the SEM. (C) At time zero, 1 μM FT₂·WT TTR was added to healthy donor plasma to initiate the subunit exchange reaction. An aliquot was immediately removed, and A2 was added at a final concentration of 30 μM (A2 at 0 h, solid green line). Both samples were incubated at 25 °C for 24 h. At 24 h, a second aliquot was removed and A2 was added at a final concentration of 30 μM and the mixture incubated at 25 °C for 3 h to allow for complete covalent modification of TTR by A2 (A2 at 24 h, dotted black line). Each sample was then analyzed by ion exchange chromatography. (D) Recombinant WT TTR (5 μM) was incubated with FT₂·WT TTR (5 μM) for 96 h. At each time point, subunit exchange was assessed by measuring Trp fluorescence (solid black line) or A2·TTR conjugate fluorescence (dotted red line). Symbols represent means; error bars represent the SEM, and lines represent fitted curves. (E) Healthy donor plasma was incubated with FT₂·WT TTR for 21 days. The sample was incubated with A2 (30 μM) and then separated by ion exchange chromatography (top panel). Peaks were collected from four identical injections, pooled, concentrated, and separated by SDS–PAGE followed by Western blotting for TTR or FLAG-tagged TTR (bottom panels). The expected pattern of WT and FT₂·WT TTR monomers is detected in peaks 1–5. Total human plasma (Plsm) was included as a control.