Limited Tryptic Digestion of Human Serum Low‐Density Lipoprotein: Islation and Characterisation of the‐Deficient Particle and of Its Apoprotien

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Abstract
Limited tryptic digestion of human serum low-density (LD) lipoprotein (.rho. [density] 1.024-1.045 g/ml) under defined conditions permitted isolation by gel filtration chromatography of a stable, protein-deficient lipoprotein; the liberated protein was separated as a mixture of peptides of low MW (< 5000). Comparison of the chemical, physical and immunological characteristics of the trypsin-treated LD-lipoprotein with those of the native preparation revealed several differences; a diminished protein content (loss of some 20-25% of the total protein of LD-lipoprotein) and increased proportions of the various lipid components, except for triglyceride (probably resulting from a loss of bound free fatty acids with the liberated peptides); a greater heterogeneity in particle size and slightly larger mean diameter; a lower hydrated density and greater peak sf [flotation] rate than the native LD-lipoprotein; an increased net negative charge; and a partial immunological identity between LD-lipoprotein and the corresponding trypsin-treated fraction. While the amino acid compositions of the protein moieties of LD-lipoprotein and of trypsin-treated LD-lipoprotein were essentially identical, trypsin-treated apo-LD-lipoprotein was distinct in its complete solubility in urea-containing buffers at high concentrations, and also in its partial solubility in buffers lacking denaturing agents. Comparison of the apoproteins of the native and trypsin-treated LD-lipoproteins by electrophoretic techniques based on MW revealed a transformation of the high MW material (> 250000) characteristic of apo-LD lipoprotein into several polypeptide species (10 major forms) ranging in size from 161,500 to about 10,000. The largest of these (band b1: 161,500) could be completely dissociated into smaller components (b2: 93,500 and b3: 77,000) upon extensive heat treatment at 90.degree. C. Electrophoresis of the soluble fraction of apo-LD-lipoprotein and of that from its trypsin-treated counterpart in polyacrylamide gels containing urea at basic pH showed the disappearance of the small amounts (< 5%) of C apoproteins of apo-LD-lipoprotein upon tryptic treatment. These results, which were highly reproducible in LD-lipoprotein preparations from different individuals, suggest that trypsin-treated LD-lipoprotein may provide a model for investigation of the organization and structural role of the principal apoprotein (apolipoprotein-B) in the LD-lipoprotein molecule.