Hypochlorite action on plasma fibronectin promotes its extended conformation in complexes with antibodies.
We investigated the influence of hypochlorite (HOCl/OCl-) on plasma fibronectin (Fn) aggregation and examined an affinity of Fn aggregates to Fn specific antibodies. Human plasma Fn HOCl/OCl(-)-mediated modification was monitored with differential OD method and with measurements of tryptophan fluorescence followed by acrylamide quenching of tryptophan emission. Antibody fibronectin complex formation was examined in ELISA systems with chemiluminescence (CL) detection. Results were expressed as an average of three experiments performed in triplicate. Fn aggregation/fragmentation was monitored with dynamic light scattering method. It was showed that HOCl/OCl- mediated chlorination promotes Fn aggregation/fragmentation with concomitant oxidation of tryptophan moieties and dichlorotyrosine formation. Quenching experiments revealed that in chlorinated Fn the percentage of intact tryptophan moieties buried in the hydrophobic Fn core increases as compared to unchlorinated Fn. In general, ELISA experiments showed that chlorination of plasma Fn diminished the number of available epitopes but for lower HOCl/OCl- concentrations (1-2 mM) the reverse effect is observed--the number of accessible fibronectin epitopes is increased when Fn adopts extended conformation in complex with antibody. Our results suggest that HOCl/OCl(-)-mediated plasma Fn chlorination leads to the formation of soluble aggregates and is followed by refolding processes. Fn chlorination with low doses of HOCl/OCl- promotes extended Fn conformation which in turn increases affinity toward specific antibodies and may promote Fn-IgG cluster formation. Thus it seems possible that mildly chlorinated plasma Fn promotes formation of IgG clusters which in turn may activate neutrophils.
Olszowski
Slawomir
S
Jagiellonian University, Collegium Medicum, Institute of Medical Biochemistry, Kopernika 7, 31-034, Kraków, Poland. mbeolszo@cyf-kr.edu.pl
Olszowska
Ewa
E
Kusior
Dorota
D
Piwowarczyk
Miroslaw
M
Stelmaszynska
Teresa
T
D020106:Acrylamide; D000906:Antibodies / Q000276:immunology*; D000936:Antigen-Antibody Complex / Q000737:chemistry* / Q000276:immunology*; D002713:Chlorine / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D005353:Fibronectins / Q000097:blood / Q000737:chemistry* / Q000276:immunology*; D005453:Fluorescence; D006801:Humans; D006997:Hypochlorous Acid / Q000737:chemistry*; D008163:Luminescent Measurements; D014364:Tryptophan / Q000032:analysis / Q000737:chemistry; D014443:Tyrosine / Q000032:analysis / Q000737:chemistry
D000906:Antibodies; D000936:Antigen-Antibody Complex; D005353:Fibronectins; D020106:Acrylamide; D014443:Tyrosine; D002713:Chlorine; D006997:Hypochlorous Acid; D014364:Tryptophan