Conformation-specific precipitation of human α2-macroglobulin by divalent zinc or calf thymus histone H3

Highly purified native α₂-macroglobulin (α₂M), α₂M-trypsin, and α₂M-methylamine were compared in experiments designed to study protein precipitation. Significant turbidity developed within 30 min in solutions containing histone H3 and either α₂M-methylamine or α₂M-trypsin, as determined by absorbance at λ = 550 nm. No turbidity was detected in solutions that contained histone H3 and native α₂M or histone H3 alone. Experiments with radioiodinated histone H3 or radioiodinated proteinase inhibitor confirmed that both the H3 and the α₂M “fast” forms (α₂M-methylamine, α₂M-trypsin) were present in the precipitates generated. As much as 70% of the ¹²⁵I-α₂M-methylamine was recovered in the precipitate after incubation with a 120-fold molar excess of H3 (concentration of α₂M-methylamine, 0.28 μm). The ratio of histone to proteinase inhibitor by weight in the precipitate was approximately two. Under comparable conditions, somewhat less α₂M-trypsin precipitated from solutions containing H3 than did α₂M-methylamine; however, inactivation of the α₂M-trypsin with phenylmethylsulfonyl fluoride prior to incubation increased the level of precipitation significantly. Solutions containing poly-l-lysine (M_r ~ 13,000) instead of histone did not form precipitates with any of the forms of α₂M studied. In a second set of experiments, radioiodinated native α₂M, α₂M-trypsin, and α₂M-methylamine were incubated in solutions containing ZnCl₂, BaCl₂, CdCl₂, CuSO₄, MgCl₂, or NiCl₂ (concentration of divalent cation between 5 μm and 1.0 mm). Native α₂M was soluble in all of these salts. By contrast, α₂M-methylamine and α₂M-trypsin precipitated extensively from solutions containing greater than 100 μm ZnCl₂. Precipitation was greater than 90% complete at 1 mm ZnCl₂. A similar effect was not observed with any of the other divalent cations.