Kinetics of triiodothyronine dissociation from bovine serum albumin: modification of the resin capture method with subsequent computer modeling.

There has been much debate about the mechanism by which thyroid hormone leaves the circulation and enters cells. Critical to the development of theoretical models of thyroid hormone uptake are precise estimates of the kinetic constants describing the reversible binding of the hormone to its binding proteins. A resin capture method was modified to permit the collection of dissociation data at intervals of 0.12 sec. Columns (30 X 0.6 cm) of Amberlite IRA 400 anion exchange resin were perfused at 30 ml/min with 3.5% dextran in modified Krebs Henseleit bicarbonate buffer at pH 7.4 and 37 C. Injection of [125I]triiodothyronine (T3) tracer in BSA-free medium established the rate constant for resin capture of free T3 in this system: kRes = 0.89 +/- 0.04 sec-1 (mean +/- SD, n = 4). [125I]T3 preequilibrated with BSA was infused to achieve final BSA concentrations of 2.3, 48, 120, 290, 507, and 725 microM, yielding apparent uptake rate constants (kapp) of 0.77 +/- 0.12 (n = 6), 0.23 +/- 0.01 (n = 4), 0.17 +/- 0.04 (n = 4), 0.057 +/- 0.005 (n = 4), 0.039 +/- 0.003 (n = 4), 0.020 +/- 0.002 (n = 4) sec-1 (SSE less than 9.4 X 10(-2), respectively, when fitted by nonlinear methods. Solving the equation kapp = kRes/(1 + [BSA]/KD') gave KD' = 1.80 +/- 0.07 X 10(-5) M (r2 = 0.96, P less than 0.001, n = 26) where KD' is the apparent or flow equilibrium constant. In a further experiment T3:BSA reassociation was eliminated by BSA dilution. This resulted in determination of the dissociation rate constant kdissoc = 0.63 +/- 0.16 sec-1 (n = 8) and therefore, the association rate constant kassoc = 3.5 X 10(4) M-1 sec-1. Mathematical modeling predicted that 14.4 microM BSA would decrease the rate of T3 uptake by the resin system by 65%. The free fraction of T3 during a single pass extraction was predicted to be greater than that measured by equilibrium dialysis, suggesting that dissociation of T3 from BSA is favored in the flux model relative to that measured at equilibrium in vitro.