title: Collagen type II modification by hypochlorite.
abstract: Oxidation of proteins is a common phenomenon in the inflammatory process mediated by highly reactive agents such as hypochlorite (HOCl/OCl(-)) produced by activated neutrophils. For instance, in rheumatoid arthritis hypochlorite plays an important role in joint destruction. One of the major targets for HOCl/OCl(-) is collagen type II (CII) - the primary cartilage protein. In our study, HOCl/OCl(-) mediated collagen II modifications were tested using various methods: circular dichroism (CD), HPLC, ELISA, dynamic light scattering (DLS), fluorimetry and spectrophotometry. It was shown that hypochlorite action causes deamination with consecutive carbonyl group formation and transformation of tyrosine residues to dichlorotyrosine. Moreover, it was shown that ammonium chloramine (NH(2)Cl) formed in the reaction mixture reacts with CII. However, in this case the yield of carbonyl groups and dichlorotyrosine is lower than that observed for HOCl/OCl(-) by 50%. CD data revealed that collagen II exists as a random coil in the samples and that chlorination is followed by CII fragmentation. In the range of low HOCl/OCl(-) concentrations (up to 1 mM) 10-90 kDa peptides are predominant whereas massive production of shorter peptides was observed for high (5 mM) hypochlorite concentration. DLS measurements showed that chlorination with HOCl/OCl(-) decreases the radius of collagen II aggregates from 30 to 6.8 nm. Taking into account the fact that chlorinated collagen is partially degraded, the DLS results suggest that smaller micelles are formed of the 10-90 kDa peptide fraction. Moreover, collagen chlorination results in epitope modification which affects CII recognition by anti-CII antibodies. Finally, since in the synovial fluid the plausible hypochlorite concentration is smaller than that used in the model the change of size of molecular aggregates seems to be the best marker of hypochlorite-mediated collagen oxidation.
authors.affiliation: Institute of Medical Chemistry, Collegium Medicum, Jagiellonian University, 31-121 Kraków, Czysta 18, Poland.
authors.forename: Sławomir
authors.initials: S
authors.lastname: Olszowski
authors.forename: Paweł
authors.initials: P
authors.lastname: Mak
authors.forename: Ewa
authors.initials: E
authors.lastname: Olszowska
authors.forename: Janusz
authors.initials: J
authors.lastname: Marcinkiewicz
mesh_terms: D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D000936:Antigen-Antibody Complex / Q000737:chemistry / Q000276:immunology; D002356:Cartilage / Q000737:chemistry; D002645:Chickens; D002713:Chlorine / Q000737:chemistry; D002942:Circular Dichroism; D024043:Collagen Type II / Q000737:chemistry* / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006997:Hypochlorous Acid / Q000737:chemistry*; D018836:Inflammation Mediators / Q000494:pharmacology; D010084:Oxidation-Reduction; D017433:Protein Structure, Secondary; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D013654:Taurine / Q000031:analogs & derivatives* / Q000494:pharmacology; D014443:Tyrosine / Q000031:analogs & derivatives
chemical_list: D000596:Amino Acids; D000936:Antigen-Antibody Complex; D024043:Collagen Type II; D018836:Inflammation Mediators; D000644:Quaternary Ammonium Compounds; D013654:Taurine; D014443:Tyrosine; D002713:Chlorine; C043410:N-chlorotaurine; D006997:Hypochlorous Acid