ИСТИНА

Cholinesterases are serine hydrolases that catalyze the hydrolysis of esters of choline, thiocholine, indole derivatives and a wide range of drugs. Both butyrylcholinesterase and acetylcholinesterase are expressed in multiple organs, including cholinergic system that plays the key role in processes such as memory, immunity regulation and vegetative organs regulation. Therefore there is a rising demand for reliable and sensitive determination of the enzymatic activity in the blood. Since thiocholine is a hydrolysis product of human cholinesterases, its accumulation can represent enzymatic status in organism. Nowadays multiple methods of analyzing thiocholine concentration are known, but the most promising method in this area is Raman spectroscopy (RS). To develop method for RS determining cholinesterase activity, the effect of different substrates, application methods and the presence of an internal standard were studied. Thiocholine was analyzed on multiple substrates: SERS-paper; CeO2 films with adsorbed gold nanoparticles through the polymer; aluminum; silver and carbon screen-printed electrodes; adsorptive immobilization of the silver nanoparticles from colloidal solution. Linear dependence of Raman lines intensity from thiocholine concentration was obtained. The detection limit of thiocholine is 200 nM that is significantly lower than the values detected by other methods. The kinetics of hydrolysis of butyrylthiocholine by butyrylcholinesterase was studied. Experimental kinetic curve fits well the published data. The effective Michaelis constant was calculated.