Denaturation and Proteolytic Digestion of Porcine Low-Density Lipoprotein in Aqueous Guanidine Hydrochloride Solutions1

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Abstract
The denaturation of porcine low-density lipoprotein (LDL) in aqueous guanidine hydrochloride (GuHCl) was studied by flotation velocity experiments, optical rotatory dispersion and fluorescence spectroscopy. The denaturation of LDL occurred between 2 and 4M GuHCl, where small sigmoidal changes in optical rotation and fluorescence intensity were noted. The hydrated density of the native LDL was 1.036g/cm3 and this remained constant upon denaturation in 4M GuHCl. The slope of the flotation coefficient-solvent density curve was 35% less for denatured LDL than for the native LDL. Since there is no indication of splitting of LDL in 4m GuHCl, it is natural to interpret the result in terms of an increase of the translational frictional coefficient by 50%. The observed changes in optical rotation, fluorescence intensity and flotation coefficient in 4M GuHCl were readily reversed and native LDL was recovered after removal of GuHCl by dialysis. Proteolytic treatment of denatured LDL produced digested LDL which had a hydrated density of 1.021g/cm3 corresponding to the loss of 30% of apo-LDL. The digested LDL behaved like a compact, globular particle in aqueous NaCl solution and in 4M GuHCl. These results can best be interpreted by a model of the LDL particle in which approximately 30% of apo-LDL is exposed to the solvent, such that it can be reversibly denatured by GuHCl and at the same time is easily available to proteolytic enzymes, whereas the rest of apo-LDL is tightly associated with lipids and possibly buried inside the lipid moiety. SDS-polyacrylamide gel electrophoresis olihe digested LDL revealed four-majot peptide fragments with, sizes ranging from 70,000 to 100,000 daltons. We believe that the method and results described in this paper will have meaningful applications in the study of membrane proteins.

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