Association-dependent active folding of alpha and beta subunits of lutropin (luteinizing hormone)

The kinetics of the structural transition of ovine lutropin (luteinizing hormone) from a dissociated and partially unfolded state to a biologically active, folded and associated state were studied from pH 1.8 to 6.5 by difference spectroscopy, circular dichroism, light scattering and ultracentrifugation under various conditions of temperature and ionic strength. Between pH 2.8 and 5.3 there is a thermodynamically reversible equilibrium between the two states of the hormone with a half-transition pH of 4.3 ± 0.1 at 26 °C. The interconversion of native folding to dissociated state is strictly first-order, and most of the kinetic results can be described in terms of two exponential decays with lifetimes of 20 and 100 seconds at pH 2 and 26 °C with one intermediate. A second intermediate with a short lifetime (1.5 s) is detected with stopped-flow experiments; its spectroscopic contribution is small. Refolding from the dissociated state is always second-order in the concentration range studied (12 to 110 μm lutropin), with rates at 26 °C, 1.4 m−1 s−1 at pH 4.3 and 2.2 m−1 s−1 at pH 5.3. The temperature dependence of the rate constant of active folding at pH 5.3 corresponds to activation parameters ΔH∗ = +23.5 kcal mol−1 and ΔS∗ = +21.6 cal deg−1 mol−1.