Acid-induced unfolding of didecameric keyhole limpet hemocyanin: detection and characterizations of decameric and tetrameric intermediate states.

Keyhole limpet hemocyanin (KLH) is widely used as an immune stimulant and hapten carrier derived from a marine mollusc Megathura crenulata. To provide details of the stability and equilibrium of KLH, different intermediate species were investigated with a series of biophysical techniques: circular dichroism, binding of hydrophobic dye, 1-anilino-8-naphthalene sulfonic acid, acrylamide-induced fluorescence quenching, thermal stability and dynamic light scattering. KLH in its native state at pH 7.4 exists in the stable didecameric form with hydrodynamic radii (Rh) of 28.22 nm, which is approximately equal to a molecular mass of 8.8+/-0.6 MDa. The experimental results demonstrated the presence of two structurally distinct species in the conformational transition of KLH under acidic conditions. One species populates at pH 2.8, characterized as decameric (4.8+/-0.2 MDa; Rh=22.02 nm), molten globule-like state, while the other accumulates at pH 1.2 and is characterized as a tetramer (2.4+/-0.8 MDa; Rh=16.47 nm) with more organized secondary and tertiary structures. Our experimental manipulation of the oligomeric states of KLH has provided data that correlate well with the known oligomeric forms obtained from total KLH formed in vivo and extends our understanding of multimer formation by KLH. The results are of particular interest in light of the important role of the mechanistic pathway of pH-dependent structural changes of Hc stability in the biochemical and medical applications of these respiratory proteins.