Monoclonal antibodies against two electron reduced riboflavin and a quantification of affinity constants for this oxygen-sensitive molecule.
In order to create a protein environment that binds preferentially to the two-electron reduced form of flavin, monoclonal antibodies have been raised against a reduced flavin derivative. Due to the low fluorescence quantum yield and visible light absorption and to the instability of reduced flavin in an aerobic environment, it is not possible to determine the affinities of these antibodies for two-electron-reduced flavin using standard techniques. Because of its sensitivity, time-resolved fluorescence can be used to overcome this problem. This technique has been applied to study the binding of two antibodies, an IgG1 and an IgM, to reduced riboflavin (1,5-dihydroriboflavin) and oxidized riboflavin (riboflavin). The affinity of the IgG1 is more than 80 times larger for 1,5-dihydroriboflavin than for riboflavin. From analysis of the dynamical parameters of the system it is apparent that the internal motion of 1,5-dihydroriboflavin bound to IgG1 is much more restricted than that of riboflavin. In contrast, the affinity of the IgM is only slightly higher for 1,5-dihydroriboflavin than for riboflavin and the flexibility of binding of both flavin redox states in the antigen binding site is almost similar.
Department of Biochemistry, Agricultural University, Wageningen, The Netherlands.
Y E
YE
Bruggeman
R G
RG
Schoenmakers
A
A
Schots
E H
EH
Pap
A
A
Van Hoek
A J
AJ
Visser
R
R
Hilhorst
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology* / Q000302:isolation & purification; D001666:Binding Sites, Antibody; D004583:Electrons; D005454:Fluorescence Polarization; D006241:Haptens / Q000096:biosynthesis; D051379:Mice; D008807:Mice, Inbred BALB C; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry; D012256:Riboflavin / Q000031:analogs & derivatives / Q000737:chemistry / Q000276:immunology*; D013050:Spectrometry, Fluorescence
D000911:Antibodies, Monoclonal; D006241:Haptens; D010100:Oxygen; D012256:Riboflavin