CS268700B2 - Means for beta-d-galacosidase determination - Google Patents

Abstract

Phenolsulphophthaleinyl beta-D-galactosides of formula I R1-R4=H, halogen, NO2 or amino; R5-R12=H, halogen, lower alkyl, lower alkoxy, OH, COOH ro NO2; M=H, alkali, alkaline earth or ammonium. Diagnostic agents for the detection of beta-galacto-sidase contg. (i) a cpd. (I) as a chromogenic substrate, (ii) a suitable buffer substance, and optionally (iii) further usual adjuvants.

Description

The invention relates to a composition for the determination of beta-D-galactosidase which contains phenolsulfonaphthaleinyl-beta-D-galactosides as the active ingredient.

igosaccharides or polysaccharides, which contain D-galactose with a beta-glycosidic bond, occur in virtually all organisms. Therefore, the corresponding beta-D-galactosidases (ЕС 3.2.1.23) are also widespread and can be detected in numerous microorganisms ^ animals and plants.

Beta-D-ga lactosidase fulfills many physiological functions in mammals: it plays an important role in the metabolism of carbohydrates, as it acts to hydrolyze lactose. In addition, beta-D-galactosidase is a key enzyme in the degradation of glycolipids, mucopolysaccharides and glycoproteins.

Due to its important physiological status, beta-D-galactosidase has been gaining importance in recent years. Thus, for example, this enzyme is used increasingly as an indicator enzyme for enzyme immunoassays (see, for example, Annals of Clinical Biochemistry 1, 221-240 (1979)).

The determination of beta-D-galactosidase activity therefore plays an increasing role. In this case, a sample containing galactosidase is mixed with a suitable beta-D-galactosidase substrate. This substrate is cleaved by the enzyme and one of the cleavage products is suitably determined. It can be determined either by the action of the enzyme liberated glycone or aglycone. Usually the released aglycone is determined. Suitable substrates are the natural lactose substrate as well as especially the chromogenic galactosides.

As a beta-D-galactosidase substrate it is in Biochem. Z., 209 (1960) described the phenyl 1-be-D-galactoside as well as its aromatic ring substituted derivatives (e.g. o-nitrophenyl-beta-D-galactoside or p-nitrophenyl-beta-D-galactoside) /. The phenol released by hydrolysis is determined photometrically in the ultraviolet region, or nitrophenols in the wavelength visible wavelength region. Also, an oxidative coupling with aminoantipyrine / Analytical Biochem may be added as an indicator reaction. 4Q, 281-11971 //.

For histochemical experiments, the naphthyl-beta-D-galactosides, for example the 1-naphthyl compound (Histochemistry 32 (199) 1973), the 6-bromo-2-naphthyl derivative (J. Biol), are mainly used. Chem. 122 (239), 1952] or naphthol-AS-BI-beta-D-galactoside (Histochemistry 3Z (89) (1973)). For visual observation, the resulting naphthols are reacted with various diazonium salts to give the corresponding azo dyes.

In addition, 5-bromo-4-chloro-indoxyl-beta-D-galactoside is known as the beta-galactosidase substrate. The indication reaction here is the oxidative dimerization of the indindoxyl formed to Indigo (Histochemistry £ 3/266 (1970)) or the coupling with a diazonium salt to the indolysylazo dyes (Nazochemistry 5Z / 323/1978).

The aforementioned methods of determination show considerable disadvantages. First, they are insensitive and, in addition, the substrates used in the assays are very poorly soluble.

Substantially more sensitive tests are obtained when galactosides are used as substrates, whose aglycone can be determined fluorometrically. As such a substrate is described, for example, in Proc. Nat. Acad. Sci. U. S. S., 1981 (1961) fluorescein-dibeta-D-galactoside. In addition, 2-naphthyl-beta-D-galactoside (Analytical Biochem) is used. 42, 275 (1971)] or 4-methyl-umbelliferyl-beta-D-galactoside (Biochem). J., 122 (525) (1967).

The disadvantage of the fluorometric determinations is that they have to be operated at considerable equipment costs. ,

Thus, there remains a need for substrates with which beta-D-galactosidase can be determined easily, quickly and reliably. It is therefore an object of the invention to satisfy this need.

CS 268700 В2

It has now been found that beta-β-galactosidase can be very sensitive to the visible region of the spectrum visually or by simple spectrophotometric instruments using sulfonaphthaleinyl-beta-O-galactosides as substrates. Furthermore, these compounds have the advantage that they are very readily soluble in water.

Accordingly, the present invention provides a composition for the determination of beta-O-galactosidase which contains the sulfonaphthalines L-β-β-O-galactosides of the formula I as an active ingredient.

(I)>

wherein R @ 5 to R @ 12 which may be the same or different, a hydrogen atom, a halogen atom, a nitro or amino group, R @ 5 to R @ ¹² which may be the same or a different hydrogen atom, a halogen atom, , C1 -C5 alkoxy, carboxyl or nitro; and

M * denotes a proton, an alkali metal, alkaline earth metal or ammonium ion.

The term halogen in the definition of the substituents R ¹ to R ¹² means fluorine, chlorine, bromine and iodine, preferably fluorine, chlorine and bromine. The term alkyl group having 1 to 5 carbon atoms means preferably alkyl having 1 to 5 carbon atoms, particularly preferably methyl and isopropyl. The term alkoxy having 1 to 5 carbon atoms is preferably alkoxy having 1 to 3 carbon atoms, with methoxy being particularly preferred.

The term alkali metal ion in the definition of H * is a lithium, sodium or potassium ion, with lithium and sodium ion being preferred. The alkaline earth ion is magnesium, calcium and barium, with calcium being preferred.

means ion +

or different, in each case denotes a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, preferably having 1 or 2 carbon atoms, or a benzyl group.

The term ammonium in the definition of M (NR ¹⁴ R ^{, S} R ¹⁶ R ¹⁷ ) wherein R ¹ to R ¹⁷ , which may be the same

The use of sulfonaphthalinyl-beta-D-galactosides as a substrate for beta-O-galactosidase leads to significantly more sensitive beta-o-galactosidase assay systems than previously known. Said novel substrates can be used to determine beta-O-galactosidase activity preferably in both the biochemical and chemical fields. They are more sensitive and have the following advantages:

a) Lower beta-o-galactosidase activities can be measured;

(b) smaller samples may be used;

The determination of beta-O-galactosidase activity can be performed in a substantially shorter time;

(d) a smaller number of samples and a more advantageous wavelength in the assay reduces the interference in the assay by other sample components.

The substrates of the present invention have been shown to be suitable for determining the beta-O-ga activity of dase of each origin, which can vary completely in their optimum pH values. Also in such cases, the formulations react with the substrates of formula I substantially more sensitively than the prior art test formulations.

Su Ifonphthaleinyl-beta-D-galactosides of the formula I are also suitable for immunoassay methods in which beta-O-galactosidase is used as an indicator enzyme whose activity must be detected after the immunological reaction has been carried out. Such immunoassay methods with enzymatic indication reactions are known in the art as. Enzymeimmunoassays.

These assay methods are for the determination of protein concentrations, lysis of arides, hormones, drugs and other low molecular weight substances in an amount of 10 x 10 ¹² mol / liter. Depending on the needs of the phase separation stages, a distinction is made between homogeneous and heterogeneous test runs. Further distribution may occur in a competitive and incompetent testing principle.

However, all test principles work with enzyme-anti gene or enzyme-antibody conjugates. The enzymatic indication reaction is common to all enzyme immunoassays. ;

The indicator enzyme suitable for this purpose is bet з-D-ga lafrosidase. The determination of beta-D-galactosidase in such enzymatic immunoassays is conveniently performed by adding a suitable beta-D-galactosidase substrate, enzymatically cleaving, and measuring photometrically in a conventional manner.

Thus, the improvement of the beta-D-galactosidase assay system also leads to considerable advantages in enzymatic immunoassays:

a / Higher sensitivity of the evidence also allows a reduction in the limit of detectable concentration, a shorter reaction time and a lower amount of samples to be used, thus reducing the interferences caused by the other components of the sample;

b) the more advantageous wavelength range in the evaluation reduces the possibility of failures in a particular reaction conduction where failure can occur due to insoluble components, for example by turbidity.

The compositions of the present invention contain, in addition to one or more substrates of formula (I), a suitable buffer system, as well as, optionally, other suitable conventional additives, such as wetting agents, stabilizers and the like. The compositions can be used in the form of solutions, such as lyophilisates or as powder mixtures, reagent tablets, or coated on an absorbent carrier.

The solution according to the present invention in the form of a solution preferably contains all the reagents required for the assay. Suitable solvents are water or a mixture of water and water-soluble organic solvents, such as methanol, ethanol, acetone or dimethylformamide. From the standpoint of durability, it may be advantageous to separate the reagents required for the assay into two or more solutions which are only mixed with one another during the assay.

For the preparation of a lyophilisate composition in a total weight of about 5 to 20 mg, preferably about 10 mg, a solution is dried which contains, in addition to all the reagents required for the test, conventional substances such as polyvinylpyrrolidone and possibly other fillers such as mannitol. , sorgit or xyllith.

The composition in the form of a powder mixture or reagent tablet can be prepared by mixing the necessary components with conventional additives and granulating them. Additives of this type are, for example, carbohydrates such as monosaccharides, oosaccharides or lysaccharides, or sugar alcohols such as mannitol sorbitol or xyllitol, or other soluble inert compounds such as polyethylene glycols or polyvinyl pyrrolidone. Powder mixtures or reagent tablets generally have a final weight of about 30 to 200 mg, preferably 50 to 80 mg. .

For the preparation of test strip compositions, an absorbent carrier, preferably filter paper, cellulose or synthetic fiber web, is impregnated with the necessary reagents commonly used in test strip production in a slightly volatile solvent such as water, methanol, ethyl alcohol or acetone. . This impregnation can be carried out in one operation, but it is often expedient to carry out the impregnation in a plurality of stages, impregnating solutions containing one component each. For example, in the first stage it can be impregnated with an aqueous solution containing buffer and other water-soluble component, and then in a second step a solution containing beta-β-galactosidase substrates. The finished test papers can be used as such or can be adhered to the handles in a known manner or are preferably inserted between the plastic backing 2 and the fine screen according to DE 21 18 45S.

The following are examples which serve to further illustrate the invention without limiting it.

The following abbreviations are used in the examples:

НЕ PES 2- (4- (2-hydroxyethyl) -1-piperazinyl) -ethanesulfonic acid;

BSA bovine serum albumin (bovine serum albumin);

Tween-20 polyoxyethylene (20) sorbitan monolaurate;

(N-tris) hydroxymethyl / methylglycine.

Three cin 'no

CS 268700 82

Example 1

Fixed! beta-β-galactosidase activity a / Preparation of solutions used:

Baztek-eufcu Hepes

100 mmol / l sodium chloride L-aspartate magnesium SSA

Tween-20 pH value (adjusted with sodium hydroxide)

154 mmol / 1 mmol / l g / l

0.5 g / l

7/3 / 36 ° С /

BcaseQ & ol.CQZlQh.l

5 mmol / l of phenolsulfonaphthalinyl-beta-β-galactoside sodium salt is dissolved in a buffer solution of the above composition. The pH is adjusted to 8.5 with sodium hydroxide at 37 ° C.

ŽSiaíQÍQÍ-ESIlSh.?

/

5 mmol / l of the sodium salt of 3,3-difluorophenolsulfonaphthalene-beta-O-galactoside are dissolved in a buffer solution of the above composition. The pH is adjusted to 7.5 at 37 ° C with sodium hydroxide.

/

5 mmol / l of the sodium salt of 3,3-dichlorophenolsulfonaphthaleinyl-beta-D-galactososide are dissolved in a buffer solution of the above composition. The pH of the solution is left at 7.3 (37 ° C).

8eaaeoioi_C3Z131s / /

In a buffer solution of the above composition was dissolved 5 mmol / 1 sodium salt 3.3 5.5 - tetrachlorophenol-3, 4 ", 5 ⁴ ', 6' - tetrabromsu fonftaleinyl L-beta-O-galactoside. The pH of the solution is left at 7.3 (37 ° C).

The substrate concentration and pH should be optimal for each substrate used. Thus, different values for the substrate concentration or different pH values of the individual reagent solutions can be consciously found.

Bszlsis.cozxoy

The commercially available beta-O-galactosidase from Escherichia coll is dissolved in the above buffer solution. The activity of this solution is about 0.08 U / ml (based on the manufacturer's data).

b / Perform measurement:

The measurement is performed photometrically at the wavelengths below.

Place 950 µl of reagent solution in a one centimeter cuvette at 37 ° C

50 µl enzyme solution. As a measure of reaction, 2 is an increase in extinction per unit of time (mext / min). It was found from the measured extinctions by dividing the reaction time. The following table shows the values found:

Table

reagent solution ' wavelength / nm / reaction / m e x t / m i n / 1 560 9 2 578 71, 3  578 123 4 578 121

Example 2

Determination of beta-D-galactosidase activity and / Preparation of solutions used

Solution-gufry

HEPES 50 mmol / l citric acid 50 mmol / l t r1tin 50 mmol / l sodium chloride 154 mmol / l Magnesium L-aspartate . 1 mmol / l BSA 10 g / l pH value / adjusted sodium hydroxide / 6,0 / 37 & easfiD & DÍ. £ QZtQK ..- l

5 mmol / l of 3,3-dimethylphenolsulfonaphthaleinyl-beta-D-galactoside are dissolved in a buffer solution of the above composition.

Eei2ínČDÍ_COííSh_-2 /

5 mmol / l of 3,3-dihydroxyphenolsulfonaphthalinyl-be-D-galactoside are dissolved in a buffer solution of the above composition.

Commercially available beta-D-galactosidases from Escherichia coli are dissolved in a buffer solution of the above composition. The activity of this solution is about 0.08 U / ml (based on the manufacturer's data).

b / Measurement:

950 µl of reagent solution was mixed at 37 ° C and 50 µl of enzyme solution in a 1-meter cuvette. After a reaction time of 10 minutes, the pH is adjusted to 10 and the extinction is measured. In the blank test, the same procedure is used except that the same amount of buffer solution is used instead of the enzyme solution. The temperature during the reaction and measurement is maintained at 37 ° C. The extinction difference is calculated from the detected extinction samples with and without enzyme. By dividing this extinction difference by the reaction time, the increase in extinction per unit time (mext / min) is obtained as a measure of reaction. The following table shows the values found for these substrates.

'' H

8 T a b u l к a CS 268700 82 reagent solution wavelength / hm / response rate / техt / mi n / 1 578 98 2 593 6

ad 3

Determination of beta-β-galactosidase activity of various origins

Common commercially available beta-D-galactosidase preparations will be used as a characteristic of their maximum activity at different levels.

These pH.

are developed by Manufacturer data:

ga Lactos1 gives for galactosidase Oalactosidase ga l aktos1 gives for beans / Jack Beans / Aspergillus nlger Escherichis coli bovine liver pH pH pH pH a / Preparation of used solutions

3.5

4/0

6.9

7.3

SSžXQh.QUÍCU

HEPES citric acid tri-in sodium chloride magnesium L-aspartate BSA

154 mmol / 1 mmol / 1 mmol / 1 nunol / 1 nunol / l

9/1

Buffer solutions of the above-described optimum composition of beta-galactosidases / 3.5 are prepared;

and hydrochloric acid according to sodium hydroxide

4.0;

, whereas

6.9; 7,3 /.

at 37 ° C pH adjustment pH is adjusted after s

Reagent solutions

In the buffer solution above, each dissolves 5 mmoL / l of said composition with different /

sodium salts 3,3 pH / 3,5, 4,0, 6,9, dihlorophenolsulfonaphthaleinyl-beta-D-galactoside

7/3 /

Enzyme solutions

Dissolving galactose goes beyond galactosidase galactosidase galactosldase with beta-O-galactose dase in a buffer solution with optimum pH:

/ Aspergillus Escherichia beef liver

Beans / ni ger col i

The activity of the solutions thus prepared is about 0.08 U / ml (based on the manufacturer's data).

Measurement:

The enzymatic reaction proceeds over a defined reaction time at pH always opt130 / µl en15 minutes

3.5

4.0

6.9

PH pH pH pH

optimal for the enzyme used. To measure, mix 1000 µl of reagent solution from the lysis solution at 37 ° C in a one-meter cuvette. After the reaction time, the pH was adjusted to 8.5 with sodium hydroxide and the extinction at 578 nm was determined.

The temperature s4 in the measurement reaction 1 is kept constant at 37 ° C

CS 268700 02 blank measurement, where 30 µl of buffer is added instead of enzyme solution · с / Evaluation

First, the difference between the measured value for the enzyme and the blank value is determined. By dividing this difference by the reaction time, the measure of reaction is the increase in extinction per time unit (mext / min).

naměř.hodn. with enzyme - measure, value No enzyme - Λ Measure values

Д measure, values

-----------—-—-------- = reaction rate / mext / min / reaction time

The measured values for the reaction of each enzyme are shown in the following table:

Table

Enzyme z pH reaction rate / mext / min / beans / 'Jack Beans / 3.5 71 Aspergillus niger AzO 112 Escherichia coli 6/9 74 beef liver 7/3 32

The above experimental results show that sulfonaphthalinyl-beta-D-galactosides are suitable as substrates for beta-galactose dase of any origin.

Example 4

Determination of free and conjugated beta-D-galactosidase activity and / Preparation of solutions used

HEPES 100 ALIGN! mmol / l sodium chloride 154 mmo1 / 1 Magnesium L-aspartate 2 mmol / l BSA. 10 9/1 W ween-20 0 , 5 g / l pH value / set sodium hydroxide / 7 3/3?

8ίϊ9ίθίθ1.Ε0112! ' /

5 mmol / l of 3,3-dichlorophenolsulfonaphthaleinyl-beta-D-galactoside sodium salt is dissolved in the above buffer solution. The pH of the buffer solution 7.3 (37 ° C) is maintained ·

Еошшух.гггШ

The commercially available beta-D-galactosidase from Escherichia coli is dissolved in the above buffer solution. The activity of this solution is less than 0.08 U / ml (based on the manufacturer's data).

A beta-D-galactosidase-anti-agent preparation is used. The preparation of such an enzyme-antibody conjugate is known, for example, as described in Biochem. Biophys. Acta 612 (40-49, 1980).

CS 268700 02

The preparation is diluted in the above buffer to achieve comparable activity to the enzyme solution described above.

b / Measurements made

The measurement is performed photometrically at 578 nm. 950 µl of the reagent solution is mixed in a 1-meter well at 37 ° C with 50 µl of enzyme solution and 50 µl of enzyme conjugate solution, respectively. The increase in extinction per unit of time (mext / min) was determined as a measure of reaction.

In response to free beta-t-galactosidase, 124 mext / min was measured; 120 mext / min was measured with beta-β-galactosidase-anti-drug conjugate.

Both measured values show that both free and conjugated beta-D-galactosidase showed a very measurable extinction difference. Accordingly, sulfonaphthaleinyl-beta-O-galactosides are equally suitable as substrates for both free beta D-galactosidase and beta-D-galactosidase conjugates.

Thus, the novel substrates can be used not only as a means for determining free beta-β-galactosidase. They are also suitable and useful in enzyme-immunoassays in which beta-D-galactosidase is used as an indicator enzyme.

Claims (2)

SUBJECT TO THE INVENTION A composition for the determination of beta-D-galactosidase, comprising one or more chromogenic substrates and a suitable buffer, characterized in that it comprises as the chromogenic substrate phenolsulfonaphthalinyl-beta-D-galactoside of the general formula Wherein R to R, which may be the same or different, are each a hydrogen atom, a halogen atom, a nitro group or an amino group, 5 12 R to R, which may be the same or different, each represent a hydrogen atom, a halogen atom, an alkyl group having 1 to 5 carbon atoms, a hydroxy group, an alkoxy group having 1 to 5 carbon atoms, a carboxyl group or a nitro group; M * denotes a proton, an alkali metal ion, an alkaline earth metal, or an ammonium ion. 2. Composition according to claim 1, characterized in that it contains wetting agents, oxidizing agents, galenic additives and / or fillers as auxiliaries.