Localization of the Active Site through Chemical Modification of the Encephalitogenic Protein

Abstract

Abstract The encephalitogenic basic protein of the myelin contains 168 amino acid residues with a calculated molecular weight of 18,200. Among these residues, there are 2 methionines and 1 tryptophan. In order to locate and identify the number of encephalitogenic determinants of this protein, chemical degradation of the methionine and modifications of the tryptophan by various specific reagents were carried out. Cleavage of the polypeptide chain at the methionyl residues was effected with cyanogen bromide. Three peptides were produced. Peptide C-1 (20 amino acid residues) was derived from the amino-terminal part of the protein, Peptide C-2 (143 residues) from the middle part, and Peptide C-3 (3 residues) from the carboxyl terminus. Only Peptide C-2 is active at the same level as the parent protein. These results are in agreement with our previous report that part of the amino-terminal segment of the protein can be removed without affecting the biological activity. The tryptophan residue was modified by the following reagents: 2-hydroxy-5-nitrobenzyl bromide, oxidation by performic acid, alkylation with 2-nitrophenylsulfenyl chloride, and formylation with formic acid saturated with HCl gas. The modified proteins, except the formylated one, were not encephalitogenic in guinea pigs. The animals did not show any neurological sign during the assay period of 30 days. The same compounds were tested with rabbits and the rabbits developed typical experimental allergic encephalomyelitis. It is concluded that there are at least two encephalitogenic determinants. One contains tryptophan, a residue essential for activity in guinea pigs, and the other is active in rabbits without the tryptophan residue. The results suggest that the encephalitogenic determinant is species-specific.