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Determination of Acetylcholine and Acetylthiocholine Using Hydroxylamine Method.
Authors: Štěpánková Šárka | Komers Karel | Zdražilová Pavla | Čegan Alexander | Ventura Karel
Year: 2004
Type of publication: ostatní - přednáška nebo poster
Name of source: YoungChem 2004
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Language Name Abstract Keywords
cze Stanovení acetylcholinu a acetylthiocholinu pomocí hydroxylaminové metody. Byla studována a testována nová metoda na sledování kinetiky enzymatické hydrolýzy acetylcholinu (ACH) a acetylthiocholinu (ATCH). Spočívá v odebírání vzorků z reakční směsi a ve stanovení aktuální koncentrace ACH, resp. ATCH jejich reakcí s hydroxylaminem a Fe3+ , přičemž vzniká hnědočervené rozpustné barvivo, které se měří spektrofotometricky. Byly stanoveny kalibrační přímky A(412nm) vs. koncentrace TCH, resp. ATCH a metoda byla testována na hydrolýzách obou substrátů pomocí acetyl- a butyrylcholinesterázy. Získané hodnoty kinetických parametrů byly porovnány s hodnotami získanými pomocí nezávislé Ellmanovy a/nebo pH-statové metody enzyme, acetylthiocholine, hydroxylamine
eng Determination of Acetylcholine and Acetylthiocholine Using Hydroxylamine Method. Acetylcholine is a physiological substrate, which acts as neuromediator of the cholinergic nerve system in the human brain. It is hydrolyzed by several forms of cholinesterases. The lack of acetylcholine is considered to be one of the most common reasons of Alzheimer?Ls disease. The enzymatic hydrolysis of acetylcholine and acetylthiocholine by cholinesterases is usually observed by pH-stat method and spectrophotometric Ellman?Ls method. In these experiments the dependencies of concentrations of products vs. time are determined. On the other hand by using hydroxylamine method the dependencies of substrates vs. time are measured. This method is based on the findings that hydroxylamine at an alkaline pH in water rapidly converts acetylcholine stechiometrically to hydroxamic acid. Hydroxamic acid then reacts with ferric ions and absorbance of ferric-acethydroxamic acid complex is measured. Reaction path: ROOC-CH3 + NH2OH ?¨ ROCNHOH + CH3OH O / \ ROCNHOH + 1/3 Fe3+ ?¨ RC Fe/3 \ / NH The dependence of substrate concentration vs. time was correlated with the Michaelis-Menten model (see fig.). The correlations were made by means of differential and integral kinetic equations describing this model. As the result the optimal values of Michaelis constant (KM) and maximum velocity (Vm) were obtained. enzyme, acetylthiocholine, hydroxylamine

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