Detection of 3-chlorinated tyrosine residues in human cells by flow cytometry

Hypochlorite is a strong oxidant, generated under pathological conditions, with the potency to introduce chlorine atom into a number of molecules. 3-Chloro- and 3,5-dichlorotyrosine are documented to be generated by this oxidant and their elevated levels were found in many diseases. Thus, we decided to check the possibility of use of FITC-conjugated antibodies for flow cytometric detection of 3-chlorotyrosine residues in human cells (A549, MCF-7, HUVEC-ST) exposed to the action of hypochlorite. Additionally, we compared the effects of chlorohydrins and N-chloroamino acids as chlorine donors. Cell fixation and permeabilization was followed by incubation with rabbit polyclonal anti-3-chlorotyrosine primary antibody and subsequent staining with goat anti-rabbit FITC-labeled secondary antibody. For antibody isotypic control, normal rabbit IgG was employed. Hypochlorite appeared to be the most efficient from the chlorocompounds analyzed in chlorotyrozine generation in all cell lines. Statistically significant increase of fluorescence corresponding to the level of 3-chlorotyrosine residues was found in cells treated with hypochlorite even at non-toxic concentrations (< 5 μM). This effect was not observed in cells exposed to the action of chlorinated amino acids or chlorohydrins. The use of anti-3-chlorotyrosine antibodies in conjunction with fluorophore-conjugated secondary antibodies analysis allows for detection of 3-chlorotyrosine residues by flow cytometry in cells treated with low doses of hypochlorite.

Research highlights
► Protein tyrosines are significantly chlorinated by HOCl.
► Chlorinated tyrosines can be detected in cells using antibodies and flow cytometry.
► Our method allows to detect chlorinated tyrosines at non-toxic concentrations of HOCl.