CS268738B1 - A method of quantitative fluorimetric determination of hydrogen peroxide and oxidase substrates - Google Patents

Abstract

The solution concerns a method of quantitative fluorimetric determination of hydrogen peroxide and oxidase substrates. This method consists in injecting a substrate sample into a continuous flow of medium. By contacting the medium with an immobilized enzyme corresponding to the respective substrate, hydrogen peroxide is produced during enzymatic oxidation of the substrate. This then comes into contact with the immobilized peroxidase. The medium contains a chemical substance which, in the presence of hydrogen peroxide and peroxidase, is capable of forming a fluorescent product. The fluorescence detected is measured by a fluorometer. The quantification itself is carried out on the basis of comparing the fluorimetric response to a determined substrate sample and the response to known standard amounts of the substance under investigation.

Description

The invention relates to a method for quantitative fluorimetric determination of hydrogen peroxide and oxidase substrates.

The development of analytical methods enabling the quantitative determination of biologically significant substances with high sensitivity is a major problem in biochemical laboratories. For the measurement of some substances that can be converted into hydrogen peroxide by the action of oxidases, fluorometry is used, in which the respective determined substrate, the corresponding oxidase, peroxidase and a chemical substance that exhibits fluorescence after an enzymatic reaction catalyzed by peroxidase are pipetted into a fluorometer cuvette. The oxidase converts the substrate into hydrogen peroxide, which in the presence of peroxidase oxidizes the substance capable of fluorescence. The magnitude of the fluorescence effect corresponds to the amount of the determined substrate. Methods of this type are described in the book M.A.Detuca (ed.), Methods in Enzymology, vol. 57, Bioluminescence and Chemiluminescence (Academic Press, New York, 1978). This method, although sensitive, has its disadvantages. The necessity of adding a new amount of enzymes for each individual measurement means a high consumption of specific enzymes. The measurement itself is slow and laborious.

These disadvantages are eliminated by the measurement method according to the invention. It employs two basic elements of novelty. The injection of the measured samples is carried out directly into a continuous flow of medium, which eliminates the need to add individual enzymes to each sample, and the enzymes used are additionally immobilized on a suitable carrier with which the flowing medium comes into contact.

The method of quantitative determination of hydrogen peroxide and oxidase substrates consists in injecting a substrate sample into a continuous flow of medium. When the medium comes into contact with an immobilized enzyme corresponding to the respective substrate, hydrogen peroxide is produced during enzymatic oxidation of the substrate. The hydrogen peroxide produced then passes through a column with immobilized peroxidase with the medium. The medium contains a chemical substance which, in the presence of hydrogen peroxide and peroxidase, is capable of forming a fluorescent product, preferably 3-/p.hydroxyphenyl/propionic acid, comovanillic acid or hydrophenylacetic acid. This substance is an electron acceptor during the enzymatic reduction of hydrogen peroxide catalyzed by peroxidase. With a suitable combination of wavelengths for excitation and emission, fluorescence can be quantitatively measured on a connected fluorimeter. Its signal is directly proportional to the concentration of hydrogen peroxide and therefore to the original substrate sample for the given immobilized oxidase. The quantification itself is performed based on a comparison of the fluorimetric response to a determined substrate sample and the response to a known standard amount of the substance under investigation.

The principle of this method of determination can be used in the field of biochemistry, medical biochemistry, physiological and pharmacological research. Typical examples of substances that can be determined in this way are glucose, choline and cholesterol.

Example

The described method was used to determine the amount of acetylcholine /ACh/ in brain tissue extracts. For this purpose, a combination of three immobilized enzymes on three consecutive columns was used: acetylcholinesterase /forms choline from acetylcholine/, choline oxidase /forms hydrogen peroxide from choline/ and peroxidase /forms a fluorescent product of the interaction between hydrogen peroxide and 3-/p _T hydroxyphenyl/-propionic acid, which was present in the flowing medium/. Samples of known amounts of Ach /20 pmol, 40 pmol, 100 pmol, etc./, and 20 µl amounts of tissue extracts were injected into the continuous flow of medium at two-minute intervals. The response of the fluorimeter to the standard amount of ACh did not change during the experiment. By comparing the response to known amounts of ACh and to tissue extracts, it was possible to calculate the concentration of ACh in individual extracts.

Claims (1)

SUBJECT OF THE INVENTION A method for quantitative fluorimetric determination of hydrogen peroxide and oxidase substrates in a medium containing a chemical substance capable of creating a fluorescent effect in the presence of hydrogen peroxide and peroxidase, characterized in that the oxidase substrate is injected into a continuous stream of medium, with which it is brought into contact with an immobilized enzyme corresponding to the respective substrate, is quantitatively converted into hydrogen peroxide, which then passes through a column with immobilized peroxidase and a fluorometer, with the fluorometer signals being directly proportional to the amount of the determined substrate or hydrogen peroxide.