This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Transthyretin (TTR) is normally a stable plasma protein. However, in cases of familial TTR-related amyloidosis (ATTR) and senile systemic amyloidosis (SSA), TTR is deposited as amyloid fibrils, leading to organ dysfunction and possibly death. The mechanism by which TTR undergoes the transition from stable, soluble precursor to insoluble amyloid fibril, and the factors that promote this process, are largely undetermined. Most models involve the dissociation of the native tetramer as the initial step. It is largely accepted that the TTR gene mutations associated with ATTR lead to the expression of variant proteins that are intrinsically unstable and prone to aggregation. It has been suggested that amyloidogenicity may be conferred to wild-type TTR (the form deposited in SSA) by chemical modification of the lone cysteine residue (Cys10). S-sulfonation and S-cysteinylation are prevalent TTR modifications physiologically, and studies have suggested their ability to modulate the stability of TTR under denaturing conditions. In the present study, we have used fluorescence detected sedimentation velocity to quantitatively determine the effect of S-sulfonate and S-cysteine on the quaternary structural stability of fluorescently conjugated recombinant TTR under nondenaturing conditions. We determined that the dissociation constant (KD) for the S-sulfonated TTR homotetramer was 12-fold lower than the free thiol homotetramer. Conversely, the KD for the S-cysteinylated homotetramer was 3.5-fold higher than the unmodified form. In addition, we can report the direct observation of tetramer stabilization by the potential therapeutic compound diflunisal. Finally, as proof of concept, we report the sedimentation of TTR in serum and the qualitative assessment of the resulting data.