Analysis of rat serum apolipoproteins by isoelectric focusing

Analytical isoelectric focusing (IEF) has been applied to the study of the apolipoprotein components of rat serum high density and very low density lipoproteins. The apolipoproteins were separated on 7.5% polyacrylamide gels containing 6.8% urea, with a pH gradient of 4-6. The middle molecular weight range apolipoproteins were identified on IEF gels by the use of apolipoproteins purified by electrophoresis on gels containing sodium dodecyl sulfate (SDS). The A-I protein focused as 4 to 5 bands from pH 5.46 to 5.82; the A-IV protein and the arginine-rich protein each focused as 4 to 6 bands from pH 5.3 1 to 5.46. The low molecular weight proteins focused from pH. 4.43 to 4.83 and are the subject of a separate communication. Comparisons of the IEF method with SDS gel electrophoresis, polyacrylamide gel electrophoresis in urea, and Sephadex chromatography are also reported. Additional studies were also carried out that tend to rule out carbamylation or incomplete unfolding of the proteins in the presence of urea as the causes of the observed heterogeneity. Supplementary key words high density lipoprotein * very low density l poprotein . SDS electrophoresis arginine-rich protein * A-I apoprotein . A-IV apoprotein . carbamylation . Sephadex chromatography Studies on the serum lipoproteins have been limited by the fact that no single analytical or preparative technique has sufficient resolving power to adequately describe the various apolipoproteins. Because of its potential for high resolution, we have made a detailed study of the technique of isoelectric focusing (1) for the analysis of rat apolipoproteins. Some isoelectric focusing studies have been carried out on apolipoproteins of human (2-9) and rhesus monkey (lo), but there have been no reported studies with the rat. In this report, we describe the general technique of gel isoelectric focusing of apolipoproteins and, in particular, the results of isoelectric focusing of the middle molecular weight polypeptides of rat serum high density and very low density lipoproteins. By this technique it was found that there is considerable polymorphism in ARP of apo VLDL and in each of the three major polypeptides in apo HDL (A-I, A-IV, and ARP). The polymorphic forms of A-IV are not separated from those of ARP. Because the separation and analysis of rat apolipoproteins have been carried out with different techniques by various investigators (e.g., 1 1 15), we have correlated the results of Sephadex gel filtration, polyacrylamide gel electrophoresis in urea, and SDS gel electrophoresis with those obtained by isoelectric focusing. Isoelectric focusing of the low molecular weight subunits is the subject of a separate report (16). MATERIALS AND METHODS