Application of Gradient Gel Electrophoresis to Evaluation of HDL Subfractions

Gradient gel electrophoresis (GGE) was used to study molecular properties of HDL subfractions. The principles of this technique is the molecular sieving utilizing a slab of continuous concave gradient (4 to 30%) of polyacrylamide gel. HDL2 (1.063<d<1.125g/ml) and HDL3 (1.125<d<1.210g/ml) prepared by sequential ultracentrifugation, were applied to GGE. HDL3 moved faster than HDL2 and the ranges of their apparent molecular weight were (1.2-2.9)×105 and (1.9-3.3)×105, respectively. HDL represents a heterogeneous class of plasma lipoproteins not only in terms of flotation properties but also in terms of their metabolic functions. In order to subfractionate HDL in connection with its metabolic functions, we subfractionated HDL2 by heparin-Sepharose affinity chromatography according to the method of Weisgraber K.H., which could isolate HDL subclasses depending on the presence or absence of Apo E. Fractions of HDL2 by heparin-Sepharose affinity chromatography were investigated by GGE. The second peak eluted by 0.095M NaCl which was considered as HDL2 with Apo E moved slower than the first peak eluted by 0.05M NaCl, which was considered as HDL2 without Apo E, indicating that HDL2 with Apo E consists of HDL2 with the larger molecular size. The apparent molecular weight of HDL2 with Apo E and without Apo E were (1.8-4.6)×105 and (1.0-3.3)×105, respectively. The present study indicated that GGE was simple, rapid and useful technique to evaluate the molecular properties of HDL subclasses.