Abstract
Trypsin digestion of human high density lipoprotein (d 1.125-1.21 g/ml) on which the lysine residues have been masked with the reversible blocking group, 2,3,4,5-tetrahydrophthallic anhydride (THPA), was found to result in the fragmentation of the apoA-I component, but not the apoA-II component of this lipoprotein particle. Approximately 50-80% of the apoA-I polypeptide was found in a lipid-free fraction, while the residual apoA-I material plus the apoA-II moiety constituted a core particle that contained most of the original lipid. Immunological analysis indicated that such fragmentation did not affect the immunoreactivity of apoA-II, but that all immunoreactivity of apoA-I was lost within the first 30 min of trypsinization. By column chromatography and electron microscopy this core particle appeared identical in size with the untrypsinized THPA-modified HDL(3) material. Size analysis of the core particle peptides suggests that not all of the A-I molecules present on the HDL(3) are trypsinized to the same extent, which indicates possible nonequivalence of these peptide chains. Analysis of the amino acid composition revealed a somewhat greater proportion of hydrophobic residues in the lipid-bound fraction than in the lipid-free fraction. Analysis of tryptophan showed that almost all of this highly hydrophobic residue was found in the lipid-bound fraction; this suggests that lipid binding occurs preferentially in the more hydrophobic domains of the A-I molecule. Incubation of the core particle with intact apoA-I, obtained from either human or bovine HDL, showed that these proteins could be incorporated to regenerate an HDL(3) of selectively altered protein composition, compared to the original lipoprotein. It is concluded that some latitude is allowable in the surface/volume relationship in lipoproteins before reorganization of the particle is required; this might, for example, provide a mechanism whereby the HDL could serve in a storage role for the C apolipoproteins in plasma.-Swaney, J. B. Selective proteolytic digestion as a method for the modification of human HDL(3) structure.
Highlights
Trypsin digestion of human high density lipoprotein (d 1.125-1.21 g/ml) on which the lysine residues have been masked with the reversible blocking group, 2,3,4,5-tetrahydrophthallic anhydride (THPA), was found to result in the fragmentation of the apoA-I component, but not the apoA-I1 component of this lipoprotein particle
Immunodiffusion analysis showed that antigenic reactivity of the trypsinized HDLS with anti-apoA-I appears to be lost within 30240 min as well, but that apoA-I1 appears to retain its immunoreactivity even after 21 hr of trypsinization
The human high density lipoproteins (HDL) particle presented a unique opportunity to perform selective enzymatic degradation of only one of the two major protein constituents of this lipoprotein while leaving the other intact. This situation arises from the fortuitous absence of arginine residues in the apoA-I1protein, allowing us to protect this protein from tryptic cleavage by a reversible modification of the lysine residues
Summary
Trypsin digestion of human high density lipoprotein (d 1.125-1.21 g/ml) on which the lysine residues have been masked with the reversible blocking group, 2,3,4,5-tetrahydrophthallic anhydride (THPA), was found to result in the fragmentation of the apoA-I component, but not the apoA-I1 component of this lipoprotein particle. 5080%of the apoA-I polypeptide was found in a lipid-free fraction, while the residual apoA-I material plus the apoA-I1 moiety constituted a core particle that contained most of the original lipid. Size analysis of the core particle peptides suggests that not all of the A-I molecules present on the HDLs are trypsinized to the same extent, which indicates possible nonequivalence of these peptide chains. Analysis of tryptophan showed that almost all of this highly hydrophobic residue was found in the lipid-bound fraction; this suggests that lipid binding occurs preferentially in the more hydrophobic domains of the A-I molecule.
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