Limited tryptic digestion of human serum low-density lipoprotein: isolation and characterisation of the protein-deficient particle and of its apoprotein.

Limited tryptic digestion of human serum low-density (LD) lipoprotein (rho 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 molecular weight (less than 5000). Comparison of the chemical, physical and immunological characteristics of the trypsin-treated LD-lipoprotein with those of the native preparation revealed several differences, including (a) 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); (b) a greater heterogeneity in particle size and slightly larger mean diameter; (c) a lower hydrated density and greater peak sf rate than the native LD-lipoprotein (d) an increased net negative charge; and (e) 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 molecular weight revealed a transformation of the high-molecular weight material (greater than 250 000) 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 degrees 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 (less than 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 organisation and structural role of the prinicipal apoprotein (apolipoprotein-B) in the LD-lipoprotein molecule.