In the human body, high density lipoprotein (HDL) has been shown to exhibit a variety of different roles. It is responsible for the transport of cholesterol, cholesteryl esters, and various other hydrophobic components, and participates in other roles such as the prevention of low density lipoprotein oxidation and possibly the innate immune response. In this dissertation, we describe a new model to classify and study mouse lipoproteins, the pressure stability of these complexes, and various compositional characteristics of mouse HDL. Moreover, we describe a way for rapid affinity purification of the core lipoproteins.
Traditional methods to isolate HDL involve the use of the ultracentrifuge, but we have grave reservations about the integrity of the HDL isolated by this method. Previous studies have indicated a loss of proteins, in particular, loss of about a third of the apoA-I from HDL upon repeated ultracentrifugation. We demonstrate that increased hydrostatic pressure may likely cause the loss or modification of larger-sized HDL but does not appear to affect the core HDL species. However, we believe that the core lipoprotein is susceptible to degradation in a centrifugal field, as we have observed the formation of less dense and denser particles from a sample containing the core lipoprotein in the analytical ultracentrifuge.
When plasma from C57BL/6J mice were run on 4–30% native gradient gels, then stained with Sudan black B, the staining pattern revealed a core lipoprotein species at approximately 240 kDa; other bands which have not been described before are also seen. Moreover, a yellow band was seen at about 180 kDa. This yellow band was transitory; it disappeared after the plasma sample had been incubated at 37°C. Above the core band, there is a long smear of lipoproteins. Then at the top, two very sharp bands appeared at about 800 kDa. Our first task in this dissertation was to describe this system in detail. When we turned to other strains of mice, we usually saw a similar pattern, and occasionally we saw bands that had shifted to different places on our gels, and sometimes we saw the presence of two major bands.
We have made use of another method for isolating HDL, namely, antibody affinity chromatography. This method works well, but the band of HDL isolated by antibody affinity chromatography is more heterogeneous in size than the HDL in the original plasma sample. Size-fractionated affinity-purified mouse HDL was characterized by sedimentation equilibrium and the main HDL species as well as two other smaller fractions were determined to be 235, 213, and 189 kDa in molecular weight. We shall have to do additional studies to see if the composition has changed, that is, if the same proteins and lipids are present on these antibody affinity-isolated lipoproteins.
A large, HDL-like particle of about 800 kDa has been detected that gives very sharp bands on our gels. This particle could be seen by Sudan black B staining, and it was readily degraded with β-mercaptoethanol, suggesting that it was composed of a number of smaller particles held together by disulfide bonds.
Finally, we characterized the staining properties of several colored fractions obtained from a commercial preparation of Sudan black B. These different colored fractions were able to stain all the lipoproteins larger than the main lipoprotein species, however, the yellow dye component was able to stain an additional smaller, lipoprotein species we believed to be the discoidal HDL.
Our results indicate that mouse HDL may be more heterogeneous in composition than originally thought. Mouse HDL exhibits a variety of molecular weights, ranging in size from 66 to approximately 800 kDa. Additionally, both ultracentrifugally and affinity chromatography isolated mouse HDL did not fully resemble the lipoprotein species present in undisturbed plasma samples. This suggests the use of 4–30% native gradient gel electrophoresis when structural or protein compositional information is crucial to the study at hand.