Characterization of the two unique human anti-flavin monoclonal immunoglobulins.
Form A of two previously described human monoclonal anti-riboflavin IgGs, the GAR [Farhangi, M. & Osserman, E. F. (1976) N. Engl. J. Med. 294, 177-183] and DOT [Merlini, G., Bruening, R., Kyle, R. & Osserman, E. F. (1990) Mol. Immunol. 27, 385-394], has been characterized in terms of binding properties and primary structure. Both forms were isolated as immunocomplexes with bound riboflavin and gave a reconstitutable apoprotein. The riboflavin-reconstituted IgGs showed a similar visible absorption spectrum, with a marked resolution of the 445-nm band and a ratio 445-nm/370-nm peaks of 1.13 for DOT and 1.19 for GAR. Both proteins bind riboflavin, FMN and FAD with a molar ratio ligand/protein of 2:1. DOT and GAR share a very similar affinity for the flavinic ligands; the Kd values for riboflavin and FMN are in the range 1 nM; that for FAD is an order of magnitude higher. DOT and GAR do not form an adduct between the nucleophilic group sulfite and the N(5) position of the flavin, and do not stabilize any flavinic semiquinone during reduction with the xantine/xantine oxidase benzylviologen system. The primary structure of fragment antigen binding (Fab) DOT and heavy-chain variable region (VH) GAR determined in the present study and that already known for the light-chain variable region (VL) GAR [Kiefer, C. R., McGuire, B. S., Osserman, E. F. & Garver, F. A. (1983) J. Immunol. 131, 1871-1875] evidenced that the two IgGs are assembled with VL and VH chains of different subgroups; a lambda III/HIII pair in GAR, and a lambda II/HI pair in DOT. Although less similar each other than to the counterparts of the same subclasses, DOT and GAR share an exclusive identity in the VH CDR3 region.
Stoppini
M
M
Dipartimento di Biochimica, Università di Pavia, Italy.
Bellotti
V
V
Negri
A
A
Merlini
G
G
Garver
F
F
Ferri
G
G
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology*; D001666:Binding Sites, Antibody; D005415:Flavins / Q000276:immunology*; D006801:Humans; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence
D000911:Antibodies, Monoclonal; D005415:Flavins