SU1571070A1 - Method of identifying lectines - Google Patents

Abstract

The invention relates to biotechnology, in particular to plant biochemistry, and can be used in plant biochemistry and pathogens and in medical biochemistry to identify components of lectins in protein spectra. The aim of the invention is to simplify and increase the resolution of the analysis. To do this, prior to fractionation, an optimal replica composition is selected, on the surface of which lectins can be immobilized, and after fractionation, a replica is attached to the separating gel, with which the lectins bind, after which the replica is immersed in a solution of erythrocytes and the bright red bands identify lectins.

Description

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The invention relates to the field of biotechnology, in particular to the biochemistry of plants and animals, and can be used in the field of biochemistry of plant immunity to pathogens and in medical biochemistry to identify the components of lectins in protein spectra after electrophoresis, isoelectric focusing or fractionation of proteins by other methods.

Lectins are proteins that specifically bind to certain carbohydrate groups, which can be part of the glycoproteins of saliva or animal blood, egg protein, etc. An important biological role is played by carbohydrate residues located on the cell surface — erythrocytes, spores of pathogenic fungi, etc. Due to their properties, lectins are involved in recognition reactions in morphogenesis and in defense reactions of animals and plants.

The aim of the invention is to simplify and increase the resolution of the analysis.

The method consists in the fact that, prior to fractionation of unpurified proteins, the replica composition is preliminarily determined for binding the detected lectins, and after fractionation, the proteins from the separating gel are transferred to the replica with which the lectins are bound, then the replica is immersed in a suspension of erythrocytes and identified lectins on the replica on the bright red bands formed by red blood cells.

The essence of the method is as follows. Lectins bind to a replica either through a specific interaction with glycoproteins or carbohydrates included in the replica, the residues of which correspond to the specificity of lectins, or due to non-specific interaction with the material.

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evil replicas such as nitrocellulose. The lectin molecules fixed on the replica surface are capable of combining with the carbohydrate residues of erythrocyte membranes with their free reaction centers. Fixing lectins on a replica allows them to hold erythrocytes sufficiently firmly, which, for example, makes it possible even to rinse the replica in physiological saline to reduce the background from the nonspecificity of the settled erythrocytes. In the absence of such fixation, mechanically stable spectra of agglutinated erythrocytes are almost impossible to obtain or at least very difficult.

PRI me R 1. Identification of wheat germ agglutinin components (AFP) in the spectra of germ proteins after isofocusing.

The embryos are separated from the wheat grain of the Bezost 1 variety. A portion of 100 mg of the embryos ground in a mortar is poured with 1 ml of 0.1 HCI. The mixture is centrifuged, the precipitate discarded.

To select the composition of the replica optimal for the lectin to be determined, several types of replicas are prepared: PAGE treated with saliva mucoproteins; PAG treated with any other type of glycoprotein; nitrocellulose membrane treated with any glycoprotein; a gel (acrylamide, agarose, or other), which includes certain carbohydrate groups; untreated nitrocellulose membrane, etc.

On the surface of the replica, 2-10 µl drops of the studied protein solution containing lectins are applied in drops. After 10-20 minutes the replicas are washed with saline and placed in a suspension of red blood cells. Under optimal conditions for carrying out the method for the lectin under study, a bright red spot appears at the site of application of the sample. In the case of ARP, the replica consisting of PAAG containing mucoproteins of saliva and treated with glutaraldehyde to bind mucoproteins with PAAG is optimal.

Isoelectric focusing (IEF) of proteins was carried out in 6% PAG 0.2 mm thick, containing 2% ampholin with a pH range of 3-10. Samples are applied to the gel using filter paper strips in a volume of 1-20 µl. A 6% PAG replica 0.2 mm thick is polymerised on a plastic substrate. The replica contains 0.9% NaCl in phosphate buffer pH 7.5. The replica is treated for a minute with human saliva mucoproteins. Then

replica put on 40 minutes on paper moistened with 10% Glukharevy aldehyde. The replica is rinsed in saline for 5-10 minutes and lightly dried to remove moisture droplets. After IEF, a gel-replica is applied to the separating gel for 10-30 minutes. Then the replica is immersed in a suspension (1-4% by volume) of rabbit erythrocytes. After 5-20 minutes on the surface

0 gel replicas appear clear bright red stripes formed by erythrocytes associated with lectins.

After separation, a separating gel is applied with a film for 20 minutes and is trypsinized to identify the components of trypsin inhibitors. The iso-points of lectins and trypsin inhibitors from wheat germ are somewhat different.

Thus, the proposed method, unlike the prototype, allows identifying the components of lectins in the spectrum of proteins and the presence of impurities of proteins with different specificity does not affect the results.

5Im р 2. Identification of the components of the lectin beans.

The choice of the optimal variant of the replica is carried out as in Example 1. For the bean lectin, the most optimal replica is the raw nitrocellulose membrane.

Conduct IEF bean lectin preparation. An untreated nitrocellulose membrane is placed on the separating gel. After the transfer of proteins due to diffusion (10–20 min), the membrane is immersed in a suspension of erythrocytes. Bean lectin components appear as bright red stripes. Under the same conditions of ARQ

0 are very weak, i.e. The sensitivity of this variant of the method is sufficient for a lectin bean and insufficient for an ARD.

Using the proposed method

5 provides in comparison with the prototype the following advantages.

Improving the reliability and resolution of the analysis in the identification of lectin components. Components

0 lectins are detected directly on the replica from the separating gel and the admixture of other proteins does not affect the results.

Multiple replicas treated with different glycoproteins or carbohydrates can be removed from a single gel to identify different types of lectins; gelatin replicas to identify proteinase inhibitors, replicas containing substrates for identifying various enzymes and their inhibitors, and the like.

Claims (1)

Simplification and increase of production-simplification and increase of resolution of analysis, reduction of its continuity of analysis, before fractionation. There is no need for proteins to pre-select cleaning lectins. On a single gel, one replica composition can be optimal, 20–30 samples5 of detectable lectins are analyzed at the same time, and after fractional extracts of proteins containing proteins from separating gel, the lectins. Identification of the components of the lek-trans on the replica, from which lectins of tins can be spent for 20-30 min, is bound back, then the replica is immersed in the IEF. erythrocyte suspension and identify Formula 10 of the lectins on a replica by a bright red polo. The method of identifying lectins by inserts formed by erythrocytes, wherein the fractionation of proteins in gels, is subjected to fractionation in order, with the aim of proteins.