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1

Collagen type II modification by hypochlorite

100%

Olszowski S., Mak P., Olszowska E., Marcinkiewicz J.

Acta Biochimica Polonica

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2003

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tom 50

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nr 2

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^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 chlorina- tion 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.

2

Hypchlorite-mediated oxidation of tyrosine dipeptides

88%

Olszowski S., Plonka P., Olszowska E., Kusior D., Niedzwiedz A.

Acta Biochimica Polonica

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2003

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tom 50

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nr Supplement 1

3

Functional states of neutrophils as suggested by whole blood chemiluminescence

76%

Zgliczynski J. M., Kwasnowska E., Stelmaszynska T., Olszowska E., Olszowski S., Knapik J. M.

Acta Biochimica Polonica

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1988

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tom 35

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nr 4

4

Oxidative modification of ovalbumin

76%

Olszowski S., Olszowska E., Stelmaszynska T., Krawczyk A., Marcinkiewicz J., Baczek N.

Acta Biochimica Polonica

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1996

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tom 43

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nr 4

Stimulated neutrophils (PMNL) are a source of the active oxygen species: O2, H2O2 and HOCl/OCT which in turn can act on proteins yielding a variety of mixed oxidation products. A system is proposed in which a model protein — ovalbumin (OVA) first undergoes chlorination by HOC1/OCT and next is oxidised by H2O2. The modification of functional groups (-NH2, -SH, -S-S-, >C=0, Tyr and Trp) in OVA was monitored as well as their accessibility to promote aggregation. Chlorination resulted in additional inter- or intra -S-S- bond formation followed by a decrease in the total sulfhydryl group content. Amino groups were oxidised to carbonyl moieties with a concomitant acidic shift of pi. Formation of chlorotyrosine at the chlorination step was confirmed and its further H202-mediated transformation to bityrosine was demonstrated. It has also been confirmed that tryptophan, and not tyrosine, is the first target for chlorination. SDS/PAGE and HPLC profiles revealed that HOCiyOCl" chlorination promotes formation of aggregates stabilised by non covalent bonds. In conclusion, we suggest that a dramatic change in the OVA molecule structure begins when the molar excess of HOC1/OC1 is about 2 per one reactive group in OVA.

Ograniczanie wyników

4 Acta Biochimica Polonica

4 Olszowska E.

4 Olszowski S.

2 Marcinkiewicz J.

2 Stelmaszynska T.

1 Baczek N.

1 Knapik J. M.

1 Krawczyk A.

1 Kusior D.

1 Kwasnowska E.

1 Mak P.

1 Niedzwiedz A.

1 Plonka P.

1 Zgliczynski J. M.

2 2003

1 1996

1 1988

Collagen type II modification by hypochlorite

Hypchlorite-mediated oxidation of tyrosine dipeptides

Functional states of neutrophils as suggested by whole blood chemiluminescence

Oxidative modification of ovalbumin