CS268737B1 - A method of quantitative luminometric determination of hydrogen peroxide and an oxidase substrate - Google Patents

Abstract

The solution concerns a method for quantitative luminometric determination of hydrogen peroxide and oxidase substrates. This method 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. This then comes into contact with an immobilized peroxidase placed in the field of view of the luminometer. The medium contains a chemical substance that exhibits luminescence when reacting with hydrogen peroxide catalyzed by peroxidase. The quantification itself is based on a comparison between the responses to an unknown substrate sample to be determined and to a known amount of the substance under investigation. These determinations can be used in the fields of biochemistry, medical biochemistry, physiology and the pharmaceutical industry.

Description

The invention relates to a method for quantitative luminometric 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. Luminescence is used to measure some substances that can be converted into hydrogen peroxide by the action of oxidases, in which the respective substrate to be determined, the corresponding oxidase, peroxidase and luminol or another electron acceptor are pipetted into the cuvette of the luminometer. The oxidase converts the substrate to hydrogen peroxide, which in the presence of peroxidase reacts with luminol to produce luminescence. The magnitude of the luminescence registered by the luminometer corresponds to the original amount of the substrate to be determined. The method and its application are described in detail in the book M.A.Detuca /ed./, Methods in Enzymology, vol. 57, Bioluminescence and Chemiluminescence (Academic Press New York, 1978/.

This method, although highly 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 substrate 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 therefore consists in injecting a sample of the substrate 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 with the medium through a column with immobilized peroxidase on a carrier that is permeable to light radiation, e.g. small glass or plastic beads, walls of a glass or plastic capillary, etc.

The medium contains a chemical substance, e.g. luminol, isoluminol, etc., which serves as an electron acceptor in the reaction with hydrogen peroxide catalyzed by peroxidase and exhibits luminescence upon oxidation. Therefore, the mobilized peroxidase is placed directly in the field of view of the luminometer.

The light signal generated during the enzymatic reduction of hydrogen peroxide is directly proportional to the concentration of hydrogen peroxide and therefore the original substrate sample for the immobilized oxidase used. The quantification itself is based on a comparison between the responses to the unknown substrate sample and to a known standard amount of the substance under investigation.

The principle of this method of determination can be used in the fields of biochemistry, medical biochemistry, physiological and pharmaceutical 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 (which converts acetylcholine to choline), choline oxidase (which converts choline to hydrogen peroxide), and peroxidase.

CS 26Θ 737 Bl /catalyzes the interaction between hydrogen peroxide and luminol present in the flowing medium, thereby producing luminescence/. Into the continuous flow of medium were injected at three-minute intervals samples of known amounts of ACh /500 pmol, 200 pmol, 100 pool etc./, and 250/ul amounts of tissue extracts. The response of the luminometer 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 luminometric determination of hydrogen peroxide and oxidase substrates in a medium containing a chemical substance which, when reacted with hydrogen peroxide, catalyzed by peroxidase, serves as an electron acceptor and exhibits luminescence during oxidation, 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 located in the field of view of the luminometer and containing immobilized peroxidase, the luminometer signals being directly proportional to the amount of the respective substrate or hydrogen peroxide.