The Conformational State of Human α2-Macroglobulin Influences Its Dissociation into Half-Molecules by Sodium Thiocyanate

Sodium thiocyanate dissociates native human α2-macroglobulin into half-molecules consisting of two disulfide-bonded subunits, when the salt concentration is equal to or exceeds 1.2m. Incubation with 1.6msodium thiocyanate for 1 h at 22°C dissociates about 90% of α2-macroglobulin into half-molecules. The half-molecules remain stable when the concentration of sodium thiocyanate is reduced to 0.2mor zero, demonstrating that reassociation does not occur under these conditions. The internal thiol esters of the half-molecules are intact because they can be exposed by treatment with methylamine or trypsin. The noncovalent interaction between the disulfide-bonded dimers is stronger in the “closed-trap” than in the “open-trap” conformation of α2-macroglobulin. The cleavage in the bait region by trypsin makes α2-macroglobulin completely stable toward dissociation, and α2-macroglobulin remains in a tetrameric state in 2.2msodium thiocyanate even when trypsin is not covalently bound to it. The increase in fluorescence with time indicates that conformational changes occur as a consequence of dissociation.