EPITOPE MAPPING OF HORSERADISH-PEROXIDASE (ISOENZYME-C)

Peptide scanning (PEPSCAN) was used to determine linear antigenic dete rminants of horseradish peroxidase isoenzyme C (HRPC). For this purpos e, we synthesized 303 overlapping hexapeptide fragments (with a step o f one amino acid residue) of the protein primary structure and studied their interactions with anti-HRPC polyclonal antisera by ELISA. Exper iments with various titers of antisera allowed us to determine linear antigenic determinants of HRPC; several such determinants were spatial ly located in regular elements of the secondary structure (alpha-helic es) found both inside and outside the protein globule. A fraction of e pitopes were located in loops and folds of the HRPC peptide chain with irregular shapes. These epitopes contained several functionally impor tant residues: Arg 38, which is part of the active site of the enzyme, as well as Phe 142 and Phe 143, which form a channel allowing aromati c substrates to reach the active site. Amino acid residues that form c alcium-binding sites or occur in the vicinity of disulfide bonds are n ot involved in these epitopes.