DE4110548A1 - Competitive binding assay for herbicides - by competition with quinone aldehyde for photo-system II receptors - Google Patents

Abstract

Detection of herbicides that interfere with the photosystem II of plants is effected by a competitive binding assay in which a quinone aldehyde (I) and the herbicide compete for binding to photosystem II receptors. The source of photosystem II receptors may be (a) reaction centres isolated from photosynthetic bacteria, e.g. Rhodobacter sphaeroides, or (b) thyalkoid prepns. isolated from green plants, e.g. spinach. (I) is a quinone nonanal, e.g. 9-(5,6-dimethoxy-3-methyl- 1,4-benzoquinon-2-yl)nonanal, 9-(1,4-benzoquinon-2-yl)nonanal or 9-(1,4-naphthoquinon-2-yl)nonanal or 9-(3-methyl-1,4-naphthoquinon-2-yl)nonanal. USE - The assay may be used, e.g., to detect contamination of drinking water by herbicides such as triazines or phenylureas.

Description

Modern agriculture relies heavily on that Use of pesticides. From the different classes of pesticides herbicides have the largest share in terms of quantity. Among the Herbicides themselves dominate Photosystem II herbi zide (PS-II herbicides) such as triazines and phenylureas. By The frequent use of PS-II derbicides occurs in many representatives this class undesirably in drinking water. It exists hence a need to easily detect herbicides.

According to the invention, a method is now proposed for this purpose which one

• a) to Photosystem II, to which quinonaldehyde is coupled, act a herbicide and coupled quinonaldehyde ver urges, and if necessary,
• b) the amount of the herbicide that has displaced quinonaldehyde, determined based on the amount of light, the luciferase in itself known manner in the presence of the displaced quinonaldehyde generated.

According to the invention, quinonaldehydes are used as they are for example in German patent application P 40 34 420.7 are described. If one according to the invention a quinonaldehydes  sets, one takes advantage of their property that the Quinone component of quinonaldehyde with Photosystem II cop peln and again displaced by herbicides. On the other hand the use of quinonaldehydes offers the possibility that to quinonaldehyde quantitatively using bioluminescent light can determine that in a conventional manner in the presence of Luciferase is generated. Through this quantitative quinonaldehyde determination can again quantitatively determine the herbicide men, which displaced the released quinonaldehyde.

It is known that bacterial luciferases in the presence of reduced flavomononucleotide (FMNH ₂ ), oxygen and long-chain aliphatic aldehydes are able to produce bioluminescent light:

In vitro, the luciferase responds to aldehydes of an aliphatic one Chain of 8 to 18 carbon atoms in length with a maxi mum at 10 to 14 carbon atoms. The very effective order The conversion of chemical energy into light energy enables the Detection of extremely low concentrations of aldehydes. On this fact is due to the fact that bacterial Lu ciferases according to the invention for an attractive detection method suitable for herbicides.

The prior art has so far only been able to show that the aforementioned generation of bioluminescent light can detect alde hyde, such as n-decanal, or FMNH ₂ or substances linked to it, for example NAD via a NADH-FMN-oxi-doreductase. Luciferases are commercially available, as are FMNH ₂ and n-decanal.

Photosystem II is in higher plants, phototrophic Bak teries and blue-green algae (cyanobacteria). The Photosystem II can also be in the form of chro in the process according to the invention  matophores, chloroplasts (organelles) or membranes of the Chlo Use roblasts (thylakoids).

With regard to the prior art, reference is made, for example, to
Kuwabara & Murata, Plant Gell Physiol., 23 (1982) 533-539 for the photosystem 11 of higher plants,
Clayton & Wang, Methods Enzymology, 23 (1971) 696-704 for the photosystem 11 phototrophic bacteria and their chromatophores,
Hirschberg et al., Z. Naturforsch., 42 C (1987) 758-761 for photosystem 11 from blue-green algae and
Nelson, J. Biol. Chem., 246 (1970) 3204-3209 for photosystem 11 in the form of chloroplasts and thylakoids.

The basis for the action of the PS-II herbicides is the composition with the quinone component (ubiquinone) of the quinonaldehyde around a common binding site (Q _B ) on the photosystem II. A conceivable generalized measurement principle of both stages of the process according to the invention can thus be described as follows. A receptor binding site (here: Q _B binding site of Photosystem II) is set with a quinonaldehyde as a ligand. In the presence of a certain amount of a herbicide, an equivalent amount of quinonaldehyde is displaced. Released quinonaldehyde is available for the luciferase, is recognized by the luciferase as aldehyde and converted as an aldehyde substrate. Depending on the amount of quinonaldehyde released, luminescent light can be detected; see. Fig. 1.

Characterization of the interaction between quinonaldehydes and bacterial luciferase

The effectiveness of quinonaldehydes against bacterial luciferase was demonstrated on several luciferases. The activity maximum with regard to the aldehyde chain length and the substrate specificity with regard to the quinone content were also determined; see. Fig. 2 and 3. Here it turned out that the most effective quinonaldehydes are just as active as the standard daldaldehyde n-decanal.

Binding of the quinonaldehydes to the Q _B binding site.
An analogue to Photosystem II of the higher plants can also be found in photosynthetic bacteria (for example Rhodobacter sphaeroides). The binding of the quinonaldehydes was demonstrated in both systems.

In the higher plants, binding studies were carried out on isolated thylakoids from spinach leaves. A given amount of radiolabelled herbicide is displaced by increasing concentrations of quinonaldehyde (see Fig. 4 and 5). Competition from the underivatized quinones (ubiquinone, naphthoquinone, benzoquinone and 2-methylnaphthoquinone) was not observed. This means that the binding constants for the quinonaldehydes are expected to be better than those of the non-derivatized quinones due to the alkyl radical.

For the bacterial system, the central part of the photosystem, the reaction center, was isolated and the proportion of internal ubiquinone removed from the Q _B binding site. Activity measurements (see experimental part) show that the quinonaldehydes bind to the reaction center and are also photochemically active. With the exception of ubiquinone (UQ ₀ ), the non-derivatized quinones are all photochemically inactive (see Table 1).

With regard to the use of bacterial luciferase in an automated analysis system, the adaptation of the reaction using a flow-through luminescence cuvette to a flow injection system (FIA) was shown (flow diagram see Fig. 6). The dependence on the flow rate was determined as shown in Fig. 7.

Table 1

Activity test on the isolated Rhodobacter sphaeroides reaction center

Fig. 1:
General scheme for the detection of ligands

Fig. 2:
Relationship between the luciferase activity and the aldehyde chain length of w- (1,4-quinon-2-yl) alkanols (NQXA) NQ9A = 9- (1,4-naphthoquinon-2-yl) nonanal
Luciferases from Photobacterium fischeri (Boehringer) and Vibrio harveyi (self-isolated)

Fig. 3:
Concentration dependence of the luciferase activity of various w- (1,4-quinon-2-yl) nonanal
UQ9A = 9- (5,6-dimethoxy-3-methyl-1,4-benzoquinon-2-yl) nonanal
BQ9A = 9- (1,4-benzoquinon-2-yl) nonanal
NQ9A = 9- (1,4-naphthoquinon-2-yl) nonanal
VitK9A = 9- (3-methyl-1,4-naphthoquinon-2-yl) nonanal

Fig. 4 + 5
Displacement of 14C-terbutryn (0.2 nmol) by w- (1,4-quinon-2-yl) nonanal

UQ9A = 9- (5,6-dimethoxy-3-methyl-1,4-benzoquinon-2-yl) nonanal
BQ9A = 9- (1,4-benzoquinon-2-yl) nonanal
NQ9A = 9- (1,4-naphthoquinon-2-yl) nonanal
VitK9A = 9- (3-methyl-1,4-naphthoquinon-2-yl) nonanal

Fig. 6:
Flow diagram of the measuring device for determining the luciferase activity in a flow injection system

Fig. 7:
Dependency of luciferase activity on the flow rate in the flow injection system
RLU / s = Relative light units per second

Experimental part Isolation of the reaction centers from Rhodobacter sphaeroides Cell disruption

The cells are in 20 mM Tris pH 8 (buffer A) with a Homogenizer homogenized. The cell suspension will then 45 minutes at room temperature with lysozyme and DNAse I incubated. Then the cells are replaced by a Ultrasound treatment in an ice bath unlocked (60 minutes step 5 continuously). It closes a 30 minute Centrifugation at 12,000 rpm (Sorvall 5534) to separate the Cell drummer. The chromatophores are in the ultracentrifuge pelletized at 100,000 xg (Ti60, 45,000 rpm) and the pellet then homogenized in buffer A.

Fractional extraction

The volume of the above suspension is adjusted to OD ₈₆₈ = 50 and fed to the first extraction. For this purpose, the suspension is brought to 125 mM NaCl, 0.5 mM PMSF and 0.25% N, N-dimethyldodecylaminooxide (LDAO) and left to stir for 45 minutes at room temperature in the dark. The chromatophores are centrifuged again as above (2 hours, 100,000 xg) and the supernatant is discarded. The pellet is taken up in the same volume as in the first extraction in buffer A and homogenized. For the second extraction, 125 mM NaCl and 0.25% LDAO are added and the mixture is stirred for 30 minutes at room temperature in the dark. The supernatant is then centrifuged again at 45,000 rpm for 2 hours with the same volume of buffer A and used for the first ion exchange chromatography.

Ion exchange chromatography

The diluted supernatant of the second extraction is made on a DE  52 column (DEAE cellulose equilibrated with buffer A, 30 × 2 cm) charged with approx. 100 ml / minute. The pillar must be with everyone Operations can be shielded from light and the insulation can at room temperature. The loaded column is washed overnight with buffer A + 0.08% LDAO and 80 mM NaCl. In the second Wash cycle, the salt concentration of the buffer to 110 mM and increased to 135 mM NaCl in the third. The elution takes place with the same buffer but 205 mM NaCl. From the eluates Spectra recorded and those containing reaction centers Fractions overnight against buffer A + 0.08% LDAO at 4 ° C in Dark dialyzed.

The second cleaning step is carried out using a MONO Q column with an FPLC system under the following conditions: -8 ml Mono Q pillar from Pharmacia

• Buffer A: 10 mM Tris pH8, 0.08% LDAO
• Buffer B: Buffer A + 200 mM NaCl
• - flow rate 3.5 ml / minute
• - Column with buffer A + 400 mM NaCl

wash, equilibrate with buffer A and loaded with protein

• - 20 ml of buffer A
• - 200 ml gradient from 0 to 100% buffer B
• - 40 ml buffer B

The elution is followed by a UV detector and the fractions containing reaction centers by absorption spectra identified.

Remove the UQ ₁₀ from the Q _B binding site and reconstitute with quinones
About 20 ml of reaction center (RC) (OD ₈₀₄ = 0.92 = 60 nmol) are added to about 2 ml of DEAE cellulose ion exchanger (DE 52 from Whatman) and incubated for 10 minutes with gentle shaking. The suspension is then placed in a column in which a small Sephadex G-200 bed has been placed. The temperature is set to 26 ° C. and the column is washed with 70 ml of 20 mM Tris pH 8/3% LDAO / 1 mM o-phenanthroline / 1 mM DTT (flow rate approx. 1.5 ml / min). This is followed by a second washing with 20 mM Tris pH 8 / 0.08% LDAO (150 ml) and then elution with 0.5 M NaCl in the same buffer. The combined eluates are dialyzed overnight against 20 mM Tris pH 8 / 0.08% LDAO (1).

For reconstitution, Q-free RC is incubated in a concentrated form (OD ₈₀₀ = 20) at the chosen concentration of the chosen quinone for 10 hours at 4 ° C in the dark.

Activity test of the reaction center from R. spaeroides

It is an absorption measurement with cross exposure (self-made).
Wavelength of the absorption measurement 550 nm,
Cross exposure wavelength <600 nm.
In order to test the activity of the reaction center of Photosystem II and the acceptor properties of the chosen quinonaldehyde, an oxidation of cytochrome C is carried out by the reaction center. The measurement is carried out under anaerobic conditions in order to prevent oxidation of the reduced cytochrome C by atmospheric oxygen. It is done by gassing with nitrogen.

1 ml reaction medium contains:

980 µl 20 mM Tris pH 7.5
10 µl 1 mM quinone in EtOH
10 µl 10 µM reaction center
12.5 µl 800 µM cytochrome c

The components are pipetted into a cuvette and after Turning on the cross exposure will decrease the absorption 550 nm tracked.

The cytochrome c is previously replaced by a small amount of sodium Dithionite reduced. The deep red solution is not allowed  turn into bright red, because under these conditions Excess sodium dithionite is present and a quantification the amount of cytochrome consumed becomes impossible.

Isolation of thylakoids from spinach leaves

Solution I:
0.4 M sucrose
20 mM tricine pH 8.0
10 mM NaCl

Solution II:
5mM tricin pH 8.5

1. Homogenize cold, washed spinach leaves without the center rib in solution I with a mixer,
2. Filter the homogenate through two layers of nylon,
3. Centrifuge the filtrate at 5000 g for 10 minutes,
4. Wash pellet with solution I and centrifuge as above,
5. Resuspend pellet in solution II (osmotic shock) and centrifuge at 17,000 g for 2 minutes,
If the thylakoids are kept in liquid nitrogen until use, the pellet is taken up in solution I with 10% glycerol (1 mg Chl / ml).

Determination of the chlorophyll content

Mix 10 ml 80% acetone with 50 µl thylakoid suspension, filter and determine the absorbance at 652 nm.

Displacement experiment on thylakoids

To each two ml of thylakoid suspension (50 µg Chl / ml in 20 mM tricine, pH 8.0, 20 mM MgCl ₂ ), identical amounts of radiolabelled herbicide are added as inhibitors. After an incubation time of 10 minutes, different concentrations of a second displacement partner are added, which may be a quinone, a quinoaldehyde or another herbicide. After another 10 minute incubation, centrifugation is carried out at 10,000 g for 10 minutes and the radioactivity in the supernatant and in the pellet is determined.

Activity determination of luciferase

The test batch has a total volume of 1 ml and continues as follows together:

885 µl buffer (0.1 M phosphate pH 6.9 / 0.2% BSA)
5 µl aldehyde in ethanol
10 µl luciferase (0.5 mg / ml)

Start the reaction by injecting 100 µl 50 µM FMNH ₂ . The integral of the resulting light quanta is measured within the first second after injection.

Measurement of luciferase activity in a flow-injection system

The structure is shown in Fig. 6. The luminescent cuvette has a measuring volume of approx. 150 µl and is provided with a photomultiplier at one end.

Since FMNH _{2 is} obtained by reduction with sodium dithionite under nitrogen, one of the two carrier strands must be oxygen-free (see competitive reaction between reduced FMN and O ₂ ). However, the whole system cannot be kept oxygen-free because the luciferase reaction itself requires oxygen. This results in the need for an oxygen-free and containing carrier stream. Both meet immediately in the cuvette. With the first injection through valve 1, 200 µl of a substrate solution (50 µM FMNH ₂ + aldehyde in 0.1 M phosphate buffer, 0.2% BSA) enters the carrier stream 1. The second injection through valve 2 of 30 µl luciferase solution is chosen in time so that it falls in the middle of the first injection.

Abbreviations:

BQ = 1,4-benzoquinone
BQ9A = 9- (1,4-benzoquinon-2-yl) nonanal
DTT = dithiothreit
FPLC = Fast Pressure Liquid Chromatography
LDAO = N, N-dimethyldodecylamine oxide
NQ = 1,4-naphthoquinone
NQXA = X- (1,4-naphthoquinon-2-yl) alkanal
PMSF = phenylmethylsulfonyl fluoride
UQ₀ = 2,3-dimethoxy-5-methyl-1,4-benzoquinone
UQ₆ = 2,3-dimethoxy-5-methyl-6-all-trans-farnesylfarnesyl-1,4-benzoquinone
UQ9A = 9- (5,6-dimethoxy-3-methyl-1,4-benzoquinon-2-yl) nonanal
UQ₁₀ = ubiquinone 50
VITK9A = 9- (3-methyl-1,4-naphthoquinon-2-yl) nonanal

Claims (6)

1. A method for the detection of substances which can be coupled to the photosystem II as ligands, characterized in that a herbicide (optionally contained in a medium) is detected using quinonaldehyde using a detection method known per se which is based on competition, taking advantage of the competition between the herbicide and the quinonaldehyde against Photosystem II. 2. The method according to claim 1, characterized in that one the amount of quinonaldehyde, that of the amount of herbicide to be detected corresponds to luciferase, determined on the basis of the amount of light in a manner known per se in the presence of the amount of herbicide corresponding amount of quinonaldehyde generated. 3. The method according to claim 1 or 2, characterized in that

• a) has a herbicide act on photosystem II, to which quinonaldehyde is coupled, and has coupled quinonaldehyde displaced and, if appropriate,
• b) the amount of the herbicide which has displaced quinonaldehyde is determined on the basis of the amount of light which luciferase produces in a manner known per se in the presence of the displaced quinonaldehyde.

4. The method according to any one of the preceding claims, characterized in that the process within the framework a flow injection analysis.   5. The method according to any one of the preceding claims, characterized in that the method with the help a biosensor and / or as a bioassay. 6. The method according to any one of the preceding claims, characterized in that the method using a Immunobiosensors and / or as homogeneous or heterogeneous Performs immunoassay.