KINETIC STUDIES OF ANTIGEN-ANTIBODY REACTION BETWEEN FRUIT BROMELAIN AND ITS UNIVALENT ANTIBODY BY MEANS OF FLUOROMETRY

概要

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The interaction of fruit bromelain with o-dansylated tyrosine residues (o-DNS fruit bromelain) and univalent anti fruit bromelain antibodies was kinetically analyzed by fluorometric measurement. The o-DNS-fruit bromelain was prepared by the following steps; 1) maleylation of fruit bromelain for masking ε-amino groups and N-terminal amino group, 2) dansylation of phenolic hydroxyl groups of maleylated fruit bromelain, and 3) denaleylation ar low pH. A univalent antibody was obtained from papain digests of the two kinds of anti-fruit bromelain antibodies, anti-fruit bromelain antibody cross-reacting with stem bromelain [IgG(cS)] and anti-fruit bromelain antibody not cross-reacting with stem bromelain [IgG(nS)] . The binding of univalent antibody to o-DNS fruit bromelain brought about the quenching of fluorescence at 495 nm emitted by excitation at 280 nm. The dissociation constants (Kd) calculated from a kinetic analysis of fluorescent quenching were 2.64×10^<-6> M for Fab(cS) [univalent antibody of IgG(cS)] and 2.49×10^<-6> M for Fab(nS)[univalent antibody of IgG(nS)], and the heterogeneity indexes of both dissociation constants were approximately 1.0. The mechanism of fluorescent quenching and the excitation energy transfer on o-DNS fruit bromelain is discussed.
名古屋市立大学の論文
1983-07-25