DD147368A1 - METHOD OF INCREASING THE ACTIVITY OF SEA-RETROSPECT PEROXIDASE - Google Patents

Abstract

A process for increasing the activity of horseradish peroxidase serves to increase the chromogen conversion. The method finds application in the field of diagnostic medicine in all detection methods in which peroxidase is turned on in a catalytic reaction. Other application examples with the same methods are chemistry, biochemistry, food chemistry, biology, immunobiology, veterinary medicine. The aim of the invention is to lower the lower detection limit for peroxidase, to increase the analytical sensitivity of this reaction or to reduce the peroxidase use with the same sensitivity. The object of the invention to increase the substrate turnover by delaying the denaturation of the enzyme during the reaction by neutral detergents is achieved by adding polyoxyethylene sorbitol ester or by polyoxyethylene octylphenol to the substrate solution. The concentration of the detergents is preferably 1 ml (= 1.11 g / l) and the reaction temperature is preferably 25 ° C, the pH being 5.0 and the reaction time being 60 to 120 min.

Description

118-1-

Process for increasing the activity of horseradish peroxide cheese

Field of application of the invention

The invention relates to a process for increasing the activity of peroxidase and is applicable wherever a peroxidase-catalyzed substrate reaction is measured. It can thus find application in clinical biochemistry _{ biochemistry _{ food chemistry, immunology, biology, pathology, pharmacology in the fields of enzymology , Histochemistry and enzyme immuno techniques _e

Peroxidase catalyzes the following reaction:

D has the function of the proton donor and is oxidized in the course of the reaction. "The proton donor is usually dyes (Chroroogene), which in the oxidized state have a higher extinction (color development or color reproduction)., It also determines the extinction coefficient of the oxidized dye on the strength of Therefore, to increase sensitivity, new proton donors were constantly being developed with irramsr larger extinction coefficients in the oxidized state, such as 4,4'-diaminodiphenyl (benzidine), 2-methoxyphenol (guaiacol), 3,3-dimethoxybenzidine (oriho- Dianisidine), ortho-phenylenediuria, 2,2-azino-di (3-ethylbenzthiazoline-sulfonate (6)) (ABTS) * '.'..- ..'

At the same time as an alternative, it was tried to optimize the reaction conditions, thereby increasing the substrate composition.

As activators for the peroxidase reaction, ammonium, cyanide ions (FRIDOVICH, 0.: 3. Biol. Chemie 238, 3921 (1963)) and manganese ions (KLAPPER, MH and HACKETT, DP: 3. Biol. Chemie 238, 3736 (1963) ) used. However, they act only at high pHs, such as pH 8 or pH 9, far beyond the optimal reaction conditions, which are at pH 5, activating using ortho-dianisidine and ortho-phenylenediamine. When using guaiacol as chromogen they are ineffective. Activation aer peroxidase under optimal reaction conditions is not known.

According to GALLATI, H .: 0. Clin. Chem. Clin. Biochem. 15: 699 (1977) and MAEHLY, A.C., in: Methods in Enzymology, New York, 1955, vol. 2, 801, is a stable complex between enzyme and Η ₂ O 'cause of increasing enzyme denaturation during the reaction and As a result, depending on the HpOp concentration, only insignificant amounts of substrate are converted after 30 to 60 minutes, and correspondingly only slight color increases are achieved.

Object of the invention

The aim of the invention is to lower the lower detection limit for peroxidase and to increase the analytical sensitivity of the method.

Explanation of the essence of the invention

The object of the invention is to increase the conversion of substrate by peroxidase by delaying the denaturation of the enzyme during the reaction by neutral detergents. This object is achieved by admixing the substrate solution with neutral detergents (such as, for example, the substrate class polyoxyethylenes as sorbitol ester or octylphenols).

Dio detergents are a Konzentrationsbsrsich of 0.01 ml / 1 to 10 ml / 1, preferably 1 ml / 1 added in liquid form with the density of _{f i li g / ml at 20 ⁰ C) of Substratlösung.in (fig _e I ) Dio temperatures in the implementation of Substratlösiing with detergents under catalytic action of peroxidase are 4 ⁰ C to 40 ⁰ C, preferably 25 ⁰ C, with a reaction time of 1 hour proved that the inactivation by neutral detergents increases with increasing reaction temperature (Fig _e 2, 3) _e The optimal reaction temperature of the peroxidase is shifted by detergents to higher temperatures at a defined reaction time and HpOg concentration (Fig. 4). The limits of the temperature increase are set to 40 C by other process-specific aspects. "The difference in activity between substrate solution alone and with addition of detergent additive increases with increasing reaction time (Abb _e 5). Here, the Inaktivierungsschutz caused by detergents results in a linear relationship between enzyme concentration and enzyme activity "Time optimally determined for a sufficient Extinktionszuwachs is 2 h _e The increase in activity is independent of the chromogen, however optimally determined Substratge differs in magnitude at όοη for each Chromogen "mix · in addition to free peroxidase is also to proteins covalently bound peroxidase activated by detergents (eg. B _e peroxidase coupled to antibodies or antigens)»

embodiments

Example 1:

Each 10 ng of purified peroxidase (adjusted by appropriate dilution) with a purity of 2.7, specific activity 535 ΙΕ / mg are dissolved in 50 ml of phosphate buffered saline pH 7.2 and react with 1.45 ml of substrate solution A, which has the following composition (serves as comparison solution):

171 IS

0.60 ml acetate buffer 1 molar pH 5.0 0.60 ml H ₂ O ₂ (66.0 mmol / 1 distilled water) 0.50 ml o-dianisidine (6 mg / 0.5 ml methanol) 58.80 ml H ₂ O

60.50 ral total volume

and 1.45 ml Substrate Solution B, composed as follows;

0.60 ml acetate buffer 1 molar pH 5.0 0.60 ml H ₂ O ₂ (66.0 mmol / l aqua dest.) 0.50 ml o-dianisidine (6 mg / 0.5 ml methanol) 58.74 ml H ₂ O.

0.06 ml of polyoxyethylene sorbitol ester

60.50 ml total volume

The reaction temperature was 20 C _# The extinction was determined every 30 minutes after the reaction stop with 10 XjI a 5n HCl on the spectrophotometer at a wavelength Λ «403 nm« The production of oxidized chromogen by peroxidase in the conversion of the substrate solution with polyoxyethylene sorbitol (solution B) after 120 minutes of reaction time, 2.7 times the conversion of the substrate mixture without detergent (solution A). Extinction measurements at the various time intervals are shown in Table 1.

Without addition of detergent, the enzyme activity, measured by the change in extinction per time interval within the second hour of reaction time, is only 11 % of the activity within the first 60 minutes. With addition of detergent, the enzyme activity within the first 60 minutes l, 9 times higher than without detergent additive and within the second hour only drops to 67 % of the one-hour value. The character. The reaction kinetics approached under the listed reaction conditions by the detergent addition of a reaction 0, order (Figure 5).

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Example 2:. ,

The 5 ng peroxidase-IgG conjugate prepared by glutaraldehyde crosslinking by the two-step method dissolved in 50 μl of phosphate buffered saline reacted with .500 All Substrate Solution A and Substrate Solution B (see Example 1). _C Dia Extinction of both batches occurred every 30 minutes after reactivity stop with lOxUl of a 5n HCl on the spectrophotometer at a wavelength A = 403 nm.

Without addition of detergent, the enzyme activity, measured by the change in absorbance per time interval within the second hour, is only 7 % of the activity within the first 60 minutes. With detergent addition, the enzyme activity is 3.2 times higher in the first 60 minutes than without detergent addition and decreases within the second hour, only 40% of Einstundenwertes _e from the absorbance of the reacted substrate solution is Detergenzienzusatz after 120 minutes of reaction time 4.2 times the absorbance of the reacted substrate solution without Detergenzienzusatz (Fig. 6) "

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Description of the pictures

Fig. 1 - Influence of the detergent concentration in the reaction mixture on the activity of the same enzyme concentration at different reaction times (reaction temperature T = 25 ⁰ C), K = control without detergents

Fig. 2 - Oxidation of chromogen (o-dianisidine) by horseradish peroxidase (1 ng peroxidase / measurement batch) as a function of the reaction time at different reaction temperatures. No addition of detergents to the reaction mixture.

Fig. 3 - Oxidation of chromogen (o-dianisidine) by horseradish peroxidase (1 ng peroxidase / measurement batch) as a function of the reaction time at different reaction temperatures. Add 0.1 ml of Tween (121 dc) to 100 ml of reaction mixture.

Fig. 4 - Oxidation of chromogen by horseradish percxidase (1 ng peroxidase / measurement batch) as a function of the reaction temperature at different reaction times.

    ο ο reaction mixture without addition of

detergents

Φ '»Reaction mixture with added

Polyoxyethylene sorbitol ester (0.1 ml / 100 ml)

_ ^ _ __ __ ___ __ connecting line of the reaction

Optima of the peroxidase in Reaktionsgeraischen without detergents and with detergents

Fig. 5 - Enzyme kinetics of equal HRP concentrations (1 ng peroxidase / assay rate)

Reaction conditions; pH word = 5.0

i-LOg concentration = 0 _# 65 mftol / 1

T = 25 ^° C.

o ~ ~ - - - ο Reaction mixture with detergents

(1 ml polyoxyethylene sorbitol ester / 1 reaction mixture).

₀ - «- ο Reaction mixture without addition of detergents

Fig. 6 - Enzyme kinetics of equal conjugate concentrations (5 ng conjugate / assay) Reaction conditions: pH = 5.0

= 0.65 mmol / l

T β 25 ⁰ C

o Reaction mixture with detergents, (1 ml polyoxyethylene sorbitol ester / l

Reaction mixture) β reaction mixture without addition of detergent

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Claims (2)

· - * - ti 711 a Fiction of the invention \ ' 1 A process for the activity enhancement of horseradish peroxidase according to known methods, in which peroxidase oxidizes both in uncoupled and in a covalently coupled to protein coupled formations in substrate solutions, characterized in that neutral detergents (density = 1.11 g / ml) in Concentrations of 0.01 ml / 1 to 10 ml / 1, preferably 1 ml / 1 are added to the substrate mixture (solution), wherein the Ρττ value is 5 »0, the reaction temperature 25 ⁰ C and the reaction time 1 h to 2 h , Method according to item 1, characterized in that polyoxyethylene sorbitol esters or polyoxyethylene-octylphenol are used as neutral detergents For this. i> Pages drawings