Characterization of the two unique human anti-flavin monoclonal immunoglobulins

M. Stoppini, V. Bellotti, A. Negri, G. Merlini, F. Garver, G. Ferri

Research output: Contribution to journalArticlepeer-review

Abstract

Form A of two previously described human monoclonal anti-riboflavin IgGs, the GAR [Farhangi, M. and Osserman, E.F. (1976) N. Engl. J. Med. 294, 177-183] and DOT [Merlini, G., Bruening, R., Kyle, R. and 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 K(d) 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 (V(H)) GAR determined in the present study and that already known for the light-chain variable region (V(L) GAR [Kiefer, C.R., McGuire, B.S., Osserman, E.F. and Garver, F.A. (1983) J. Immunol. 131, 1871-1875] evidenced that the two IgGs are assembled with V(L) and V(H) chains of different subgroups; a λIII/HIII pair in GAR, and a λII/HI pair in DOT. Although less similar to each other than to the counterparts of the same subclasses, DOT and GAR share an exclusive identity in the V(H) CDR3 region.

Original languageEnglish
Pages (from-to)886-893
Number of pages8
JournalEuropean Journal of Biochemistry
Volume228
Issue number3
DOIs
Publication statusPublished - 1995

Keywords

  • anti-flavin IgG
  • human monoclonal immunoglobulins
  • primary structure

ASJC Scopus subject areas

  • Biochemistry

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