Ammosova T N, Ouporov I V, Ignatenko O V, Egorov A M, Kolesanova E F, Archakov A I
Department of Chemical Enzymology, School of Chemistry, Lomonosov Moscow State University, Russia.
Biochemistry (Mosc). 1997 Apr;62(4):440-7.
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.
肽扫描（PEPSCAN）用于确定辣根过氧化物酶同工酶C（HRPC）的线性抗原决定簇。为此，我们合成了该蛋白质一级结构的303个重叠六肽片段（步长为一个氨基酸残基），并通过酶联免疫吸附测定法（ELISA）研究了它们与抗HRPC多克隆抗血清的相互作用。使用不同滴度抗血清进行的实验使我们能够确定HRPC的线性抗原决定簇；其中几个这样的决定簇在蛋白质球体内外发现的二级结构（α螺旋）的规则元件中呈空间定位。一部分表位位于HRPC肽链形状不规则的环和折叠中。这些表位包含几个功能重要的残基：作为酶活性位点一部分的精氨酸38，以及形成允许芳香底物到达活性位点通道的苯丙氨酸142和苯丙氨酸143。形成钙结合位点或出现在二硫键附近的氨基酸残基不参与这些表位。
