Epitope Mapping of Horseradish Peroxidase (Isoenzyme C)

T. N. Ammosova,^1,2 I. V. Ouporov,¹ M. Yu. Rubtsova,¹ O. V. Ignatenko,¹ A. M. Egorov,¹ E. F. Kolesanova,³ and A. I. Archakov³

¹Department of Chemical Enzymology, School of Chemistry, Lomonosov Moscow State University, Moscow, 119899 Russia; fax: (7-095) 939-27-42.

²To whom correspondence should be addressed.

³Institute of Biomedical Chemistry, Russian Academy of Medical Sciences, ul. Pogodinskaya 10, Moscow, 119832 Russia; fax: (7-095) 245-08-57.

Submitted January 30, 1997.

Peptide scanning (PEPSCAN) was used to determine linear antigenic determinants of horseradish peroxidase isoenzyme C (HRPC). For this purpose, we synthesized 303 overlapping hexapeptide fragments (with a step of one amino acid residue) of the protein primary structure and studied their interactions with anti-HRPC polyclonal antisera by ELISA. Experiments with various titers of antisera allowed us to determine linear antigenic determinants of HRPC; several such determinants were spatially located in regular elements of the secondary structure (alpha-helices) found both inside and outside the protein globule. A fraction of epitopes were located in loops and folds of the HRPC peptide chain with irregular shapes. These epitopes contained several functionally important 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 aromatic substrates to reach the active site. Amino acid residues that form calcium-binding sites or occur in the vicinity of disulfide bonds are not involved in these epitopes.

KEY WORDS: epitope mapping, horseradish peroxidase isoenzyme C, solid-phase synthesis of polypeptides, peptide scanning, homologous alignment of amino acid sequences of peroxidases.

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