Analysis of human apolipoproteins C by isoelectric focusing in immobilized pH gradients

Apolipoproteins C are involved in many ways in the metabolism of plasma lipoproteins. Apolipoproteins C from the delipidated VLDL of 35 controls and 165 normo- and hyperlipoproteinemic patients were analyzed by isoelectric focusing on an immobilized pH gradient, pH 4.0-5.0, with 7 M urea, which raised the apparent pH range to 4.8-5.7. This method is an improvement over conventional isoelectric focusing with carrier ampholytes with regard to both resolution and reproducibility. Due to the high resolution (0.1 pH units per cm) additional apolipoprotein C-III bands: C-III0 A1, C-III0 A2, C-III1 C and C-III2 C (the designations A, anodic, and C, cathodic, refer to direction of migration on IEF in relation to the main band) are described for the first time. The possible artifactual nature of these protein bands could be excluded. Cleavage with neuraminidase and peptidases, immunological detection and/or two-dimensional electrophoresis were used to obtain more information. The additional bands seem, in part, to be hydrolysis products of carboxypeptidase A (C-III1 C, C-III2 C). The appearance of C-III1 C and C-III2C was dependent upon the serum triglyceride concentration. The percent distribution of C apolipoproteins in very low density lipoproteins (VLDL) from control serum agreed with previously published data. Apolipoproteins C can also be focused in immobilized pH gradients from VLDL and serum without delipidation.