There is a lack of data about influence of lead compounds on the heat shock proteins in the fish organism. But these proteins belong to the most informative markers of toxic intoxication and general stress of the organism. Such investigations are really topical and important for estimation of the fish health during water biocenosis monitoring.

The main subject of the article was to investigate the toxic influence of lead ions on various stress-dependent biochemical systems in different tissues and organs of the scaly carp. In the scope of this aim level of the heat-shock protein Hsp60 under the action of the various concentrations of lead ions.

Scaly carps with weight 270…350 g were used for investigations. All experiments were performed in pools with volume 200 liters. Each experimental group consisted of 7 animals. Influence of the 0,2; 0,5 and 5 mg/dm3 of lead ions, that corresponds to the 2, 5 and 50 highest non-toxic dosages, were used in experiments. Fish were incubated in the media with lead ions for 96 hours, after that they were decapitated and blood and some tissues were collected for further investigations. Control group of the animals was incubated in the same media without lead ions. In the incubation media constant temperature about 18–20°С were maintained. The blood was collected from heart by Pasteur pipette. Also brain and gills were collected for experiments. All organs were washed few times in phosphate buffered saline for rests of blood removal.

Three ul of the lysate with 5.1 ug of the total protein was applied on the nitrocellulose membrane (Millipor). For the background fluorescence identification lysis buffer with the same volume as sample was used. Membrane was incubated with antibodies against heat shock proteins SAB4501464 (Sigma, USA), [5A5] (ab2787) (Abcam, USA) and [1B5] (ab19136) (Abcam, USA) in phosphate buffer solution (PBS) for 90 min. After that membrane was incubated for 30 min. with polyclonal goat anti-rabbit antibodies conjugated with alkaline phosphatase (Tropix, USA) – 1:5000 in PBS. Detection of immune complexes was performed by using a commercial substrate solution for alkaline phosphatase – CDP-Star (Tropix, UK). Visualization was done by using X-ray film ECL HyperFilm (Amersham, USA) and a set for films developing (Kodak). Images were processed using the software package GelPro (Version 3.1, USA).

Level of the heat shock protein Hsp60 was investigated by using the dot-blot hybridization method based on the interaction between specific monoclonal anti-Hsp60 antibodies with the target protein. Statistic results processing was done by using Student’s t-test with Statistik software.

It was found that in the leucocytes of the scaly carp the level of the Hsp60 dramatically increased after lead ions treatment. It should be mentioned that such increasing had dosage-dependent manner.

Similar results were obtained during investigation of lead ions influence on the Hsp60 level in brain and gills of the experimental animals. It was shown that level of the target protein increased in both organs after incubation with 0,2; 0,5 and 5 mg/dm3 of the lead ions. Highest level of the target protein was found in organs of the animals treated with 5 mg/dm3 of lead ions.

As the conclusion we can postulate that all used concentration of lead ions caused significant increase of the heat-shock protein Hsp60 that can be a good evidence of its role as stress-response factor which acts as a response of the organism to the toxic lead action.

Also our investigations promote usage of the Hsp60 as specific marker of lead toxic influence on the organism of the animals.

Keywords: heat shock proteins, HSP 60, the dot-blot analysis, scaly carp, lead

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