Protein-protein and protein-lipid interactions in human serum high-density lipoprotein: an analysis by a spin label method

The protein (apo HDL₂) from human serum high density lipoprotein of d 1.063–1.125 g/ml (HDL₂) and its major polypeptide chains III and IV, were spin-labelled with a nitroxide mixed-anhydride reagent. The electron spin resonance (E.S.R.) spectra of the labelled materials showed a dependence on pH, ionic strength and temperature. Under conditions favouring protein aggregation (isoelectric point region, high ionic strength) there was a marked accentuation of the strongly (broad signal) over the weakly (narrow signal) constrained spin label. A similar accentuation was observed in the spectra of spin-labelled apo HDL₂, or its fractions III and IV, re-lipidated with a mixture of phosphatidyl choline and cholesterol esters. Such spectra were similar to those obtained with native HDL₂, spin-labelled in its protein moiety. A systematic analysis of the graded re-lipidation of apo HDL₂ by non-polar and polar lipids showed that the spin label method employed was a good indicator of protein-lipid interactions, particularly if applied to well characterized lipid-protein complexes isolated in the ultracentrifuge. The method employed, however, provided no information on the nature of such interactions.