Evaluation of the conformational equilibrium of reduced hen egg lysozyme by antibodies to the native form.
To evaluate the conformation of reduced HEL, the monoclonal antibodies HyC1 and HyC2, which recognize different conformational epitopes on native hen egg lysozyme (HEL), were used, and the kinetics of their interactions with native HEL, S-1,2-dicarboxyethylated HEL (DCE-HEL), and carboxymethylated Cys6 and Cys127 HEL (CM(6,127)-HEL) were assessed using surface plasmon resonance. Although their association rate constants differed 10(5)-fold, their dissociation rate constants were essentially the same, suggesting that DCE-HEL and CM(6,127)-HEL possess conformations similar to that of native HEL when they bind antibodies. We considered that the ratio of the association rate constant of reduced HEL to native HEL represents the proportion of the native format determinant in equilibrium. Reduction of the Cys6-Cys127 disulfide bond would transform the epitope recognized by HyC1 into a non-native conformation similar to that of DCE-HEL. We show that monoclonal antibodies provide a sensitive tool for evaluation of the structural and hydrodynamic changes of proteins.
Oda
Masayuki
M
Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, 1-5 Hangi-cho, Shimogamo, Sakyo-ku, Kyoto 606-8522, Japan.
Kitai
Aki
A
Murakami
Akikazu
A
Nishimura
Miyuki
M
Ohkuri
Takatoshi
T
Abe
Yoshito
Y
Ueda
Tadashi
T
Nakamura
Haruki
H
Azuma
Takachika
T
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000939:Epitopes / Q000276:immunology*; D007700:Kinetics; D009113:Muramidase / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism; D010084:Oxidation-Reduction; D011487:Protein Conformation; D020673:Protein Renaturation; D012996:Solutions; D020349:Surface Plasmon Resonance
D000911:Antibodies, Monoclonal; D000939:Epitopes; D012996:Solutions; C421864:hen egg lysozyme; D009113:Muramidase