Epitope mapping of horseradish peroxidase (isoenzyme C).
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.
Ammosova
T N
TN
Department of Chemical Enzymology, School of Chemistry, Lomonosov Moscow State University, Russia.
Ouporov
I V
IV
Rubtsova MYu
Ignatenko
O V
OV
Egorov
A M
AM
Kolesanova
E F
EF
Archakov
A I
AI
D000595:Amino Acid Sequence; D001665:Binding Sites; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D006735:Horseradish Peroxidase / Q000737:chemistry* / Q000276:immunology; D007106:Immune Sera / Q000276:immunology; D007527:Isoenzymes / Q000737:chemistry / Q000276:immunology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000276:immunology; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment
D000939:Epitopes; D007106:Immune Sera; D007527:Isoenzymes; D010446:Peptide Fragments; D006735:Horseradish Peroxidase