MECHANISM OF SURAMIN-INDUCED DEOLIGOMERIZATION OF TUMOR-NECROSIS-FACTOR-ALPHA

Deoligomerization of human tumor necrosis factor alpha (TNF), spiked w ith I-125-labeled form, was studied quantitatively using size-exclusio n chromatography and off-line monitoring with a gamma-counter. A detai led investigation of the oligomeric state of TNF was carried out as a function of its own concentration (0.3-7500 nM referred to the subunit , M(r) 17 000) in the absence or in the presence of various amounts (1 0, 100, 1000 mu M) Of suramin, an inhibitor of TNF biological activity in vitro, which promotes TNF deoligomerization. The dependence of tri meric form content on total TNF concentration was modeled with a seque ntial dissociation process (trimer --> dimer --> monomer) assuming an identical dissociation constant for each step, K-dl = 0.2 nM. This mod el was used as the simplest for data fitting although, generally, no c hromatographic resolution of dimeric species could be obtained. Best f itting of all data could be achieved with a model including a conforma tional change of TNF trimer into a state more prone to deoligomerizati on (K-d2 = 400 nM), which was favored by suramin binding. A kinetic st udy of TNF dissociation by the same method produced values for the deo ligomerization rate of trimer: on the average, k(off) approximate to 4 x 10(-5) s(-1) (t(1/2) approximate to 5 h) between 4 and 20 degrees C with little dependence on suramin concentration; at 37 degrees C, a s izable increase is observed in the presence of 1 mM suramin (k(off) 2. 3 x 10(-4) s(-1), t(1/2) = 0.8 h). Data of suramin inhibition on TNF r eceptor binding, as obtained after incubation times much shorter than the above half-life of trimer, indicate that suramin binding to TNF tr imer is the early mechanism of receptor binding inhibition.