Primary structure of the C-terminal cyanogen bromide fragments II, III and IV from bovine brain proteolipid-apoprotein.

Purified lipophilin from bovine brain white matter was reductively carboxymethylated and then cleaved by cyanogen bromide into four fragments: CNBr I 18-19 kDa, CNBr II 5 kDa, CNBr III 2.1 kDa and CNBr IV 0.7 kDa. Hydrogenbromide/dimethylsulfoxide and 3-bromo-2-(2-nitrophenylsulfenyl)skatol (BNPS-skatol) cleaved lipophilin into four fragments of molecular masses of approximately 14000 (Trp I), 2100 (Trp II), 5000 (Trp III) and 7000 Da (Trp IV). Separation and purification of the peptides for liquid phase sequenator degradation was achieved by high performance liquid chromatography. In addition proteolytic cleavage of the Trp IV fragment with trypsin facilitated the alignment of the peptides. An effective control of the sequenator data came from the partial acid hydrolysis of the Trp IV fragment, which yielded di-, tri- and tetrapeptides. The mixture was N-trifluoroacetylated, the amide (peptide) bonds and carboxyl groups were reduced with B2D6 (hexadeuterodiborane) and the polyaminoalcohols derivatized with chlorotrimethylsilane. These derivatives were separated and identified by capillary gas-liquid chromatography/mass spectrometry. Extensively overlapping sequences support the data obtained by Edman degradation in a liquid phase sequenator of the CNBr peptides II, III and IV, the 72 amino acid residues containing C-terminal sequence of lipophilin of molecular mass (7520 + x) Da, which includes the Trp IV fragment.