XXII. BIOCHEMICKÝ ZJAZD - Jesseniova lekárska fakulta

Posters
59.
OPTIMALIZATION OF ELLMAN’S ASSAY TO STUDY
THE KINETICS OF CHOLINESTEraSES
Dominika Neuschlová and Anna Hrabovská
Dpt. of pharmacology and toxicology, Faculty of Pharmacy, Comenius University,
Odbojárov 10, 832 32 Bratislava
Ellman’s assay (EA) has been widely used in experimental research and clinical practice.
The limitations of this method are however a high background in biological samples,
instability of the dissolved substrate (thiocholine) and its sensitivity to the light exposure.
The aim of this project was to determine the favorable conditions for EA in order
to lower the background and the reagent instability and thus allow detecting even very
low activities of cholinesterases.
Human butyrylcholinesterase was chosen to study the conditions of EA. Butyrylthiocholine
iodide was used as a substrate. Phosphate, HEPES and Ringer buffers were used at pH
values 7,0; 7,5; 8,0; and 8,5. Stability of the substrate dissolved in each buffer was tested
over the time. The full spectrum was followed in each buffer. Velocity of the color product
production was followed as a function of time (v/t curve) and substrate concentration (v/s).
The substrate was the most stable in the presence of HEPES buffer. The charts of full
spectrums and the velocity dependences suggested the same kinetics of butyrylcholinesterase-catalyzed
reaction of butyrylthiocholine in both phosphate and HEPES buffers.
Based on our results we can conclude that Ellman’s assay performed in HEPES buffer, in
contrary to phosphate buffer, is more suitable for measuring of cholinesterase activity.
This is due to the lower background (raising from substrate instability) and unaffected
kinetics.
Acknowledgement: The project was supported by APVV grant SK-CZ-0028-09.
180 XXII. Biochemistry Congress, Martin