NO880278L - PROCEDURE FOR DETERMINING LYSOSYM ACTIVITY. - Google Patents

Description

US-PS 3,834,991 describes a colorimetric determination method for lysozyme. Whole cells of Micrococcus lysodeikticus are used and treated with reactive dye. The stained cells are incubated in aqueous solution with an undetermined amount of lysozyme. Due to cell lysis, the bound dye dissolves in the aqueous solution. The color of the solution is an approximate measure of the lysozyme concentration in the sample.

The process of cell lysis is complex and is only an indirect measure of lysozyme activity [N. Grossowicz et al., I: Methods of Biochemical Analysis 29, 435 (1983)]. It has now been shown that the test is not specific for lysozyme. When using whole cells, dyes in principle bind to free NHg or OH groups of e.g. proteins and polysaccharides in general, and teichonic acids of the cell surface. It follows that, for example, in protease-containing lysozyme samples, the protease activity is also measured as proteins are broken down. You thus get a false answer about the lysozyme concentration.

The invention therefore provides a method by which this disadvantage can be overcome. When isolated cell walls of Gram-positive bacteria are used for this sample, the exact N-acetylmuramidase activity of a sample can be calculated due to the coloring of the sample solution.

The invention thus relates to a method for determining N-acetylmuramidase activity in a sample using reactive dyes, the method being characterized by using isolated colored peptidoglycan from gram-positive bacteria as substrate.

In the following, the invention is described in detail, especially in its preferred embodiments. Furthermore, the invention is defined in the claims.

With the test method according to the invention, the N-acetylmuramidase activity of all corresponding enzyme products can be determined in principle. Preferably, however, it is used for the determination of lysozyme, especially in body fluids, e.g. human body fluids, and murine-lysing enzyme products and bacteria, especially streptomycetes.

Peptidoglycan from gram-positive bacteria, preferably from Micrococcus lysodeikticus, is used for the sample. The peptidoglycan, also called murein, is extracted according to known methods by treating the cells with trichloroacetic acid and a protease and subsequent extraction of the murein as free as possible of teichonic acids, proteins and polysaccharides. The term "freed as much as possible" means that the peptidoglysan must at least be purified to the extent of the substances mentioned, that when using N-acetylmuramidase contaminated with corresponding decomposing enzymes, one does not get any false answers about the N-acetylmuramidase activity.

Isolated peptidoglycan can be colored with reactive dyes as described, for example, in US-PS 3 934 991, 2 820 785, 2 889 316, 2 891 941, 2 892 828, 2 979 498, 3 054 795, 3 036 058, 3 149 100 or 3 127 232. These are essentially reactive dyes as they are used for dyeing cellulose-containing textiles. The dyes reactive Blue 19 (CI. No. 61200 ), CI. Reactive Green 14 ((<R>) Remazol Brilliant Grtln DGG) as well as (<R>) Cibacron Grtin 4G for the samples.

The coloring can be carried out in different ways by mixing peptidoglycan with the dye in a suitable reaction medium, for example aqueous alkali hydroxide or -carbonate resp. dilute acids, such as formic or acetic acid. The ratio in which the peptidoglycan of the dye mixture is used can vary within wide ranges, preferably, however, the two components are mixed with each other in a range from 10:1 to 1:10, preferably 1:1 to 1:3. The reaction volumes can amount to 25-100 ml per murein, preferably 50 to 100 ml. Reaction time and temperature have not been precisely determined either. Advantageously, the reaction mixture is incubated over a period of 1-20 hours at a temperature of 20 to 100°C. However, an incubation of 5 minutes at 90°C to 100°C and then 15 to 20 hours at 20°C is preferred. After the reaction has ended, the unconsumed dye is washed out with buffers of pH 3-9, such as e.g. sodium acetate or phosphate and organic solvents such as ethanol or ethers. After drying, the colored peptidoglycan can then be used for the sample.

Determination of the lysozyme activity takes place by incubation of the stained murine with the N-acetylmuramidase-containing sample. Let, for example, should be incubated 1-2 mg/ml murein with approx. 1-10 mg/l (35-350 U/ml) N-acetylmuramidase.

Here again, the reaction temperature and reaction time can fluctuate within wide ranges. It is advantageous to work at temperatures between 20 and 37°C by incubating under gentle shaking for approx. 15-60 minutes. It is particularly preferred to end the reaction after 30 minutes at 25°C. This can take place by adding, for example, alkali hydroxide [when using e.g. Reactive Blue 19 (CI. No. 61200 )] or hydrochloric acid [when using e.g. "Cibacron" green 4G or "Remazol-Brillant green GGL]. The insoluble peptidoglycan is separated by centrifugation and the extinction is measured at a wavelength suitable for the respective dye. The extinction values formed are directly proportional to the N-acetylmuramidase activity.

The invention shall be explained in more detail by means of some examples, in which the percentages refer to weight if nothing else is stated.

Examples.

1. Isolation of murein from the cell wall of Micrococcus lysodeicticus.

The method listed below is a modification of the method of Schleifer et al., Bacteriol. Rev..36, 407

(1972).

18 g of lyophilized cells of Micrococcus lysodeikticus (Sigma) are suspended in 10 liters of ice-cooled distilled water. 2.5 liters of an ice-cold 25% strength aqueous dichloroacetic acid solution are added to this suspension and incubated for 45 minutes under ice cooling. Then centrifuge at approx. 30,000 x g in a flow-through rotor (SS34, DuPont), the centrifuge is washed with 2 liters of distilled water, and resuspended again in 12.6 liters 75$

(v/v) ethanol. After stirring for 15 minutes at room temperature, centrifuge again, the centrifuged is washed with 2 liters of distilled water, taken up again in 9 liters of distilled water, and incubated at 90°C in a water bath. 2.5 liters of the 25% aqueous trichloroacetic acid are added and further incubated for 20 minutes at 90°C. The mixture is then kept in an ice bath for 15 minutes, and then centrifuged. The sediment is washed twice with 2 liters of distilled water, and suspended in 3.2 liters of 0.05 molar amino carbonate buffer (pH 7.5, 37°C). The suspension is mixed with 360 ml of 1 mg/ml trypsin in 0.05 molar ammonium carbonate buffer (pH 7.5), incubated for 2 hours at 37°C, centrifuged and washed twice with 2 liters of ice-cold distilled water. The pellet is suspended in 150 ml of distilled water and lyophilized. You get 4 g of the isolated murein.

2. Dyeing of moray eel.

1 g of lyophilized murein according to examples 1 is treated for 5 minutes in a boiling water bath with 50 ml of a solution of 2% (v/v) Reactive Blue 19 (CI. No. 61200) in 1N aqueous sodium hydroxide solution and allowed to stand overnight at room temperature. The stained murein is centrifuged for 10 minutes at 30,000 x g. The unbound dye is removed from the sediment by washing at 35 °C, respectively -50 ral water 0.08 molar sodium acetate buffer (pH 4.4), and approximately 8 times with 0, 1 molar sodium acetate buffer (pH 5), until the supernatant remaining after centrifugation is colourless. After each wash, the cells are centrifuged at 30,000 x g for 10 minutes. The colored murein is finally washed with each time 50 ml of water and ethanol and ether, and dried overnight at room temperature. The dried murein is suspended in 50 ml of distilled water, homogenized for 2 minutes with Ultraturrax and then lyophilized. You get a fine powder that can be stored for a long time at room temperature.

3. Quantitative spectrophotometric determination of lysozyme.

2 mg of the stained murein is suspended in a centrifuge tube in 0.95 ml of 0.ml N sodium acetate (pH 5.0). 50 µl of the lysozyme-containing sample (20-200 mg/l, i.e. 700-7000 U/ml) is added and incubated in a shaker at 25°C for exactly 30 minutes. The reaction is terminated by the addition of 1 ml IN aqueous sodium hydroxide solution. The mixture is centrifuged for 5 minutes at 13,000 x g.

The above extinction is determined at a wavelength of 591 nm. A blank value without lysozyme and standard in an amount of lysozyme is treated similarly as indicated above. The color of the above is directly proportional to the lysozyme concentration in the samples. The concentrations can be calculated using the standard values or read from a corresponding adjustment curve.

4. Automated spectrophotometric lysozyme test.

For the routine determination of a majority of samples, the lysozyme test mentioned in example 3 can be automated. For this, a suspension of 2 mg/ml of the colored murine is prepared in 0.1 molar sodium acetate (pH 5.0). Respectively 150 µl of this suspension is transferred by means of an automatic pipette ("Cetus Propette" Systems, Fa. Cetus, Emeryville, USA) into the 96 wells of a microtiter plate. To this is added 50 µl of a suitable diluted lysozyme sample (4-40 mg/l, i.e. 140-1400 U/ml). The plates are incubated for 30 minutes at 25° C in a shaker. The reaction is terminated by the addition of 100 µl, 4N NaOH. After the mixture is centrifuged for 15 minutes at 500 x g. Each time 200 µl of the above is transferred into a new microtiter plate and the extinction is determined at 570 nm. In each microtiter plate, the standard solution with known lysozyme concentrations is treated in the same way. An empty plate serves as a dummy value. The color of the sample supernatant is directly proportional to the lysozyme concentration present. From the adjustment values, the respective lysozyme concentration of a sample can be determined. 5. Activity determination of bacteria-lysing enzyme product.

To 2.8 ml of a suspension of 0.2 mg micrococcus luteus ATCC 4698 (Boehringer Mannheim) per ml of 0.1 molar sodium acetate (pH 5.0), 0.2 ml of bacteria-lysing enzyme product-containing samples are pipetted, and at 25° C the decrease in turbidity is determined by measuring the extinction at 450 nm. 1 U is defined as the extinction decrease of 0.001 photometric scale units per minute.

Claims (7)

1. Method for determining N-acetylmuramidase activity using reactive dyes, characterized in that lysolated colored peptidoglycan from gram-positive bacteria is used as substrate. 2. Method according to claim 1, characterized in that peptidoglycan from Micrococcus lysodeikticus is used. 3. Method according to claim 1 or 2, characterized in that peptidoglycan is used which is stained with reactive Blue 19 (CI. No. 61200), CI. Reactive Green 14 ("Remazol Brillant Grun GGL) as well as "Cibacron" Grtln 4G. 4. Method according to one or more of claims 1-3, characterized in that the colored peptidoglycan is incubated for 15-60 minutes at 20-37°C with the N-acetyl-muramidase-containing samples. 5. Method according to claim 4, characterized in that it is incubated for 30 minutes at 25°C. 6. Method according to one or more of claims 1-5, characterized in that the lysozyme activity or the N-acetylmuramidase activity of a Streptomycet enzyme product is determined. 7. Method according to one or more of claims 1-6, characterized in that the lysozyme activity or the N-acetylmuramidase activity of the human enzyme product is determined.