Characteristics of two intermediates trapped in the unfolding pathway of arginine kinase induced by guanidinium chloride

Equilibrium guanidinium chloride (GdmCI)-induced unfolding of arginine kinase (AK) was investigated by enzymatic activity, intrinsic fluorescence, 8-anilinonaphthalene-1-sulfonic acid (ANS) fluorescence, circular dichroism (CD) spectrum, and size-exclusion chromatography. The measurements showed that AK unfolded through two equilibrium intermediates: the molten globule state and the partly folded state. Both intermediates have no enzyme activity. The molten globule state exists at 0.4–0.8 mol/L GdmCI, perhaps after the N-terminal domain has unfolded but the C-terminal domain is still intact. The partly folded state occurs at 1.1–1.5 mol/L GdmCI with a hydrodynamic volume no more than 1.6-fold larger than the native state and a pronounced far UV-CD signal. Its ANS fluorescence intensity is about 50% of the molten globule state. This partly folded state shares similarities with the “burst” kinetic intermediate of protein folding.