Human Plasma High-density Lipoproteins are Stabilized by Kinetic Factors

High-density lipoproteins (HDL) are heterogeneous complexes of proteins and lipids that mediate cholesterol removal from the body. Our thermal and chemical denaturation studies of mature spherical HDL isolated from human plasma show that, contrary to the widely held assumption, the particle stability has a kinetic rather than thermodynamic origin. Guanidinum hydrochloride (GdmHCl) concentration jumps at 25 °C monitored by circular dichroism (CD) at 222 nm reveal two dominant irreversible kinetic phases in HDL denaturation. The slower phase (relaxation time τ1∼2×104 seconds) is observed in 1–6 M GdmHCl, and the faster phase (τ2∼2×103 seconds) is detected in 3–6 M GdmHCl. Comparison of the free energy barriers associated with these phases, ΔG∗=16–17 kcal mol−1, with the near-zero apparent thermodynamic stability inferred from the spectroscopic measurements after prolonged incubation in 0–6 M GdmHCl at 22 °C indicates the kinetic origin for HDL stabilization. Electron microscopic analysis of HDL incubated in 0–6 M GdmHCl suggests that the slower kinetic phase involves HDL fusion, while the faster phase involves particle rupture and release of the apolar lipid core. Thermal denaturation experiments indicate high enthalpic barriers for the particle rupture that may arise from the transient disruption of lipid and/or protein packing interactions. These results corroborate our earlier analysis of model discoidal HDL and indicate that a kinetic mechanism provides a universal natural strategy for lipoprotein stabilization. Such a mechanism may facilitate structural integrity of the heterogeneous lipoprotein particles, slow their spontaneous interconversions, and thereby modulate lipoprotein lifetime and functions.