Reactivity of monoclonal antibodies against a tryptophan-riboflavin adduct toward irradiated and non-irradiated bovine-eye-lens protein fractions: an indicator of in vivo visible-light-mediated phototransformations.
We describe here the reactivity toward the soluble protein of bovine eye lens of anti-tryptophan-riboflavin (anti-Trp-RF) adduct monoclonal antibodies, which recognize the hapten tryptophan-riboflavin generated by irradiation of a solution of bovine serum albumin in the presence of riboflavin. It is demonstrated that five different anti-Trp-RF adduct monoclonal antibodies, all belonging to the IgG1 isotype, react with the total soluble proteins of bovine eye lens. The components of the soluble protein are separated by Sephadex G-200 chromatography and the isolated fractions analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). All the separated protein fractions also react by a direct ELISA with the monoclonal antibodies; this reaction is more intense when the isolated fractions have been previously irradiated with visible light in the presence of riboflavin under an atmosphere of oxygen or nitrogen. Irradiation of the total soluble protein with visible light in the presence of riboflavin produces the appearance of new bands, corresponding to compounds of higher molecular weight. Riboflavin-sensitized irradiation of the protein fractions with visible light under an oxygen or nitrogen atmosphere is accompanied by a concomitant decrease of the tryptophan fluorescence. It is postulated that the action of visible light in the presence of either the endogenous riboflavin or its derivatives could be partly responsible for the protein aggregation observed during aging.
Mancini
M
M
Departamento de Química Física, Facultad de Química, Pontificia Universidad Católica de Chile, Santiago.
Edwards
A M
AM
Becker
M I
MI
de Ioannes
A
A
Silva
E
E
D000818:Animals; D000911:Antibodies, Monoclonal; D002417:Cattle; D002850:Chromatography, Gel; D003459:Crystallins / Q000737:chemistry* / Q000276:immunology / Q000528:radiation effects*; D004797:Enzyme-Linked Immunosorbent Assay; D007908:Lens, Crystalline / Q000737:chemistry; D008027:Light; D008956:Models, Chemical; D010777:Photochemistry; D012256:Riboflavin / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D013053:Spectrophotometry; D014364:Tryptophan / Q000032:analysis* / Q000737:chemistry / Q000276:immunology
D000911:Antibodies, Monoclonal; D003459:Crystallins; D014364:Tryptophan; D012256:Riboflavin