WO2005014147A2

WO2005014147A2 - Method for isolating metal cofactors out from biological-organic systems involving the use of preparative native continuous polyacrylamide gel electrophoresis (pnc-page)

Info

Publication number: WO2005014147A2
Application number: PCT/DE2004/001514
Authority: WO
Inventors: Bernd Kastenholz
Original assignee: Forschungszentrum Jülich GmbH
Priority date: 2003-08-05
Filing date: 2004-07-13
Publication date: 2005-02-17
Also published as: DE10336540B4; WO2005014147A3; DE10336540A1

Abstract

The invention relates to a method for isolating metal cofactors out from biological-organic systems involving the use of preparative native continuous polyacrylamide gel electrophoresis (PNC-PAGE). The invention is characterized in that an acrylamide gel with a total monomer proportion of 4 % (w/v) is used, an electrophoresis buffer having a pH value of 10.00 is used, and 0.5 µl TEMED/ml gel and 5µl APS/ml gel are used, whereby the gel is completely polymerized over a period of time of 69 hours.

Description

Process for isolating metal cofactors from biological-organic systems using preparative native continuous polyacrylamide gel electrophoresis (PNC-PAGE)

The invention relates to a method for isolating metal cofactors from biological-organic systems using preparative native continuous (PNC) polyacrylamide gel electrophoresis (PAGE).

Protein and enzyme research is of paramount importance in the context of current scientific discussions. For example, in the areas of pharmaceutical, medical, biochemical and biological basic research, questions often arise that attempt to clarify the relationship between the structure and function of proteins and enzymes in biological systems.

The development and application of methods or composite processes that come from the field of analytical chemistry play a decisive role in solving these life science questions. To assess the importance of a protein or enzyme derived from a plant, animal, human or microorganism, the protein must first be purified and isolated using analytical methods. This process must not go hand in hand with a chemical change in the protein structure. The elementary composition of the molecule must also be preserved, otherwise no valid statement can be made with regard to the function of a protein.

In this context, the preparative purification and isolation of proteins from biological-organic samples is particularly problematic. In the case of solid biomatrices, such as, for. B. plants, the soluble fractions must first be extracted from this matrix by cell disruption in an aqueous medium. The biological activity of the molecules is preserved through sample cooling.

After separation of the solid and liquid fractions, e.g. B. by centrifugation in a cooling centrifuge, the dissolved substances, which are in the cytosol, are stored over liquid nitrogen in order to exclude a chemical change of the present species and to ensure permanent preservation.

The cytosols are usually first subjected to a chromatographic purification step. Ideally, gel permeation chromatography (GPC) is used for this purpose. The sample is separated according to molecular mass. As a further step, element contents are determined in the individual GPC fractions using element-analytical methods (for example atomic absorption spectrometry) in order to make a statement about the distribution of individual elements via the detected to reach ten molecular mass ranges (element species analysis). Frequently elute in the high molecular weight range (> 10 kDa) after a GPC protein that contains metals, for example. This analytical step is very important because in the realm of proteins there are enzymes whose active center is a metal ion. The qualitative determination of these metal cofactors and their stoichiometry represents an important step in the classification of metal enzymes and metal proteins.

After a GPC, it is important to use another dimension to separate molecules to purify and isolate a particular protein. If a protein has been detected in a high molecular weight fraction with the highest concentration of a certain metal, it is possible to further separate this fraction with the help of preparative native continuous (PNC) polyacrylamide gel electrophoresis (PAGE).

Since proteins are amphoteric compounds, the charged groups in the molecule are arranged differently at different isoelectric points. With PNC-PAGE, the molecules are separated according to their isoelectric points. Native means that the proteins in this process may neither be changed structurally nor in their elemental composition. Continuously refers to the buffer system used in the PNC-PAGE. This means that both the cathode and the ano- the buffers have the same composition and the same pH value. The same buffer is also used to make the gel.

An analytical method is known from the prior art [1] which is concerned with the purification and characterization of high-molecular forms of cadmium from plant foods. After a GPC of the plant cytosols, high-molecular cadmium species (HM-Cd-SP), which had a molecular weight of approximately 200 kDa, were detected in the plants. It was found that these cadmium species are proteins.

In order to reliably rule out a change in the high molecular weight cadmium species after the GPC, no further concentration steps are usually carried out before electrophoretic purification of these substances, e.g. B. by ultrafiltration or lyophilization of the corresponding GPC fractions. Possible contamination or changes in the existing species balance due to further analytical steps are also excluded.

The PNC-PAGE method is generally used to purify the high molecular weight cadmium species after charging. This can be carried out, for example, using the "Model 491 Prep Cell" (U.S. patent number 4,877,510) from Bio Rad [2,3].

The high molecular weight cadmium bond forms separated with the PNC-PAGE were always on the same Detected in the electropherogram, however, the method had the disadvantage that the metal cofactors or cadmium concentrations in these fractions were so low that the results of these experiments were classified as semi-quantitative [1].

The object of the invention is therefore to develop a new PNC-PAGE method compared to the prior art, which makes it possible to isolate proteins containing cofactor from biological-organic systems. In particular, cofactors from the group of cadmium, zinc, palladium, iron, copper, nickel, cobalt, manganese, chromium, molybdenum and platinum are to be isolated.

With the present method it is now possible to bind low molecular weight (<10 kDa) bound and high molecular weight (> 10 kDa) bound metal cofactors from biological-organic systems, such as. B. from plant cells, animal cells, human cells or cells of microorganisms. The metal cofactors to be detected remain stable with the method according to the invention and can be eluted almost completely.

Starting from the preamble of claim 1, the object is achieved according to the invention with the features specified in the characterizing part of claim 1.

The advantageous embodiment of the acrylamide gel with a total monomer content of 4% (w = weight / v = volume) according to claim 1 causes proteins up to a molecular weight of approx. 200 kDa can move freely through the gel and be separated according to their respective isoelectric point.

The further advantageous embodiment of the electrophoresis buffer with a pH of 10.00 according to claim 1 has the effect that all proteins with isoelectric points below 10.00 (pl <10.00) are negatively charged and migrate to the anode.

The advantageous period of 69 hours for the polymerization of the gel according to claim 1 means that no reactive monomers are present in the gel during the electrophoretic purification of the proteins. This prevents the metal cofactors of the biological-organic systems to be examined from undergoing a chemical reaction with residual monomers or other substances involved in the reaction in the gel, which can lead to denaturation of the proteins.

By weighting the sample to be examined with glycerol with a volume fraction of 10% (v = volume / v = volume) based on the total volume of the biological-organic system used according to claim 2, an optimized sample compression is achieved, which leads to an improved resolution of Protein bands leads in the PNC-PAGE according to the invention. For example, the peak width of individual substances in the electropherogram is minimized.

Further advantageous developments are specified in the subclaims. The method according to the invention is to be described below by way of example.

With the PNC-PAGE method, a. the composition of the electrophoresis gel and the polymerization time of this gel are important [4].

The following chemicals were used to prepare 40 mL of a gel with a total monomer content of 4% (w / v) (= 4% T):

4 mL acrylamide / bis stock solution (40%, [g / 100mL], acrylamide / bis = 37.5: 1)

4 mL Tris-HCl buffer stock solution (200 mmol / L Tris, pH 10.00; 10 mmol / L NaN ₃ ) and 32 mL ultrapure water,

20 μL TEMED as a catalyst and

200 μL APS (10%) as a starter.

All chemicals have at the time of gel manufacture

Room temperature and are homogenized in a beaker with a magnetic stirrer for 1 minute at low speed. The APS solution (0.1 g ammonium persulfate dissolved in 1 mL water) is freshly prepared.

After pouring the gel to a height of 4 cm, the gel solution is covered with an airtight layer of 3 mL 2-propanol for 60 minutes. After about an hour, the isopropanol is stripped off and the gel surface is rinsed with 8 × 4 ml of Tris-HCl buffer (pH 10.00; 20 mM Tris; 1 mM NaN ₃ ) and then covered with a layer of 4 ml of this buffer. Under these conditions, the gel was completely polymerized at room temperature over a period of 69h00 " Siert. The electrophoretic lead was then carried out immediately.

Since the gels are subjected to high thermal loads in all PAGE tests, they are cast again for each test under the conditions mentioned. The gel column diameter is, for example, 28 mm.

For example, a Tris-HCl buffer (20 mM Tris, 1 mM NaN ₃ , pH 10.00) can be selected as the electrophoresis buffer of the continuous buffer system. The electrolyte concentration is selected so that the proteins do not have to be used to conduct the current. The volume of the cathode buffer is 500 mL, that of the anode buffer is 2000 mL.

The same buffer (20 mM Tris, 1 mM NaN ₃ , pH 8.00), V = 700 mL can be used as the elution buffer as in the prior art [1].

For example, the "Model 491 Prep Cell" from Bio Rad with its components, consisting of the upper and lower buffer chamber, the cooling finger, the gel column, the elution chamber with a dialysis membrane (molecule exclusion: 6000 Da) and the gel pouring stand can be used for electrophoresis become.

Furthermore, a DC voltage source (e.g. PowerPAC 1000, BioRad), a peristaltic pump (e.g. Model EP-1 Econo Pump, BioRad), a fraction collector (e.g. Model 2110, BioRad), a UV -Detector (e.g. Model EM-1 Econo UV Monitor, BioRad), a recorder (e.g. Model 1327 Econo Recorder, BioRad) and an um- roller pump (e.g. Buffer Recirculation Pump, BioRad). The system should be cooled, e.g. B. in a refrigerator at 4 ° C.

The following parameters were set as an example:

- Peristaltic pump: flow rate of the eluent (1 mL / min); Fraction size (5 mL); Number of fractions (80); Total volume of eluent (480 mL); Wait volume (80 mL)

- Detector: AUFS (1.0) absorption unit fill scale; λ = 254 nm - DC voltage source: Constant Power (5 W); Time (8 h)

Recorder: Range (100 mV); Paper Speed Dial (6 cm / h)

Circulation pump: flow rate (95 mL / min)

- sample (2.7 mL + 0.3 mL glycerin)

The electrophoretic lead can last for example 80 minutes. After 75 minutes of the lead, the electrophoretic process can be interrupted by turning off the power. The sample can now be carefully underlaid within 5 minutes. When the 80 minute limit is reached, the actual electrophoretic purification of the proteins can be started.

With the help of the newly developed PNC-PAGE method, more than 90% of a high-molecular cadmium species could be found for the first time after electrophoretic purification of a plant cytosol. This is an indication that these types of cadmium bonds to be separated are stable in this system and can be almost completely eluted. Furthermore, a chromatographic purification of the PAGE fraction with the highest cadmium content showed that these cadmium species elute after a GPC in the molecular weight range of 200 kDa. This also speaks for the stability of the high-molecular cadmium species to be investigated when separated using the PNC-PAGE method according to the invention.

After a GPC of a plant cytosol, high molecular weight cadmium proteins eluting in the molecular mass range of approximately 200 kDa were further purified using the PNC-PAGE method according to the invention. The result showed that these high molecular weight cadmium species could be eluted in a few fractions in the electropherogram in a peak separated from the baseline and tapering. This result has been reproduced. It suggests that the high molecular weight cadmium species is a specific cadmium protein.

After separating plant cytosols on a GPC column, certain element species are already very good highly purified. Thus, after applying a first separation according to molecular size, positive results are obtained in the structural elucidation of certain substances. Since proteins are not only found as a monomer, but also as polymeric proteins in biological samples, it is postulated that e.g. B. the high molecular weight cadmium species after a GPC as polymers (* 200 kDa). After a further separation of the corresponding GPC fraction with the PNC-PAGE, these species are detected as a monomer («67 kDa). In this case, the high molecular weight cadmium species would be a trimer that has a different isoelectric point (pl) compared to the corresponding monomer.

Both in the sample preparation of the plants described in the literature [1] and in the subsequent purification steps for isolating metal proteins, the species equilibria present in each case can be strongly influenced by the respective buffer and the pH value. For this reason, plant extraction and the purification methods used are carried out with a buffer (pH 8.00), the pH of which is close to the natural pH within a plant in the slightly alkaline pH range [6]. However, changing the pH during root extraction z. B. in the range of pH 7 to 9, the metal species also change significantly in their elution behavior after purification with HPLC [7].

The method according to the invention represents a very sensitive analytical system. can be used, for example, for the analysis of high molecular weight forms of cadmium from plant samples which behave stably under the given conditions mentioned and in particular at a pH of 10.00.

With a precise knowledge of the existing element binding forms of the metal cofactors in biological-organic systems, the development of new types of medication for the treatment of poisoning and serious diseases is possible. In addition to carrying out a chemical synthesis, the corresponding active ingredients can also be obtained from medicinal plants, algae, fungi and other organisms. PNC-PAGE therefore plays a very important role, particularly when isolating drugs from phytosystems.

For example, by complexing the metal ions by adding z. B. complexing agents inactivated enzymes and thus their effects in the organism are blocked or regulated. After a chemical reaction of the complex or chelating agent with the metal

The cofactor of the corresponding enzyme transports the complexed metal species from the cell to the organ's excretory organs. The remaining apoenzyme is denatured. A suitable chelating agent already used in metal poisoning is "Berliner Blau". This substance is, for example, able to complex thallium ions in vivo, which are then further excreted via the gastrointestinal tract. Günther and Kastenholz have an overview of the speciation of thallium in book contributions [8 ] and zinc [9] in clinical samples. The biochemical behavior and the effect of elements in biological organisms are determined by the present element species. Therefore, elemental species analysis of clinical matrices plays an important role in the differential diagnosis of diseases.

The qualitative determination of the metal cofactor and its stoichiometry represents an important step in the classification of metal enzymes and metal proteins [10]. Equally important in PNC-PAGE is the question of whether, after the purification and isolation of an enzyme, not only a certain metal ion is bound by this substance, but whether other metals can also be present in the active center of a molecule.

It is therefore sensible to carry out multi-element species analysis for the method according to the invention using PNC-PAGE in the individual PNC-PAGE fractions of the purified samples.

Coming z. B. in nickel fractions of zinc or other metals whose concentrations are above the previously determined blank value concentrations, the probability increases that in addition to nickel other elements exist as co-factors of the isolated protein-metal complex.

Furthermore, when determining the form of elective delivery in the various matrices, it must be taken into account that certain elements are ubiquitous in the environment. For this reason, care must be taken during all analytical steps to ensure that no contamination is entered into the various systems. In all processes of an analysis run there is a risk that organic and inorganic species are irreversibly changed by changing the pH value in a sample. If buffers are used, the buffer substances can interact with the sample.

To rule this out, it is important for the PNC-PAGE to check the properties of the buffers used. For example, substances exist that e.g. B. stabilize the pH of aqueous frozen enzyme solutions. A buffer system that maintains enzyme activity under these conditions is, for example, a HEPES buffer (N-2-hydroxyethylpiperazine-N ^{^} - 2-ethanesulfonic acid).

In order to carry out a precise elucidation of the structure of metal enzymes after isolation with the PNC-PAGE, it is necessary to crystallize the metal species which are in a specific PNC-PAGE fraction.

The three-dimensional structures of protein-cofactor complexes can then be elucidated, for example, by X-ray crystallography [11]. The availability of well-ordered three-dimensional crystals is a prerequisite for high-resolution X-ray structure analysis. It is the only method to gain detailed knowledge of the structure of large biological macromolecules [12]. Examples:

1. Isolation of an Iron Cofactor from Plant Cells Medically active enzymes are to be isolated from plant cells. The leaves of the plant are prepared according to the method known to the person skilled in the art and the resulting cytosols are stored in polypropylene tubes in cryoracks in a container for storing samples at low temperatures [5].

After the cytosols have been purified on a GPC column with a separation range of 20 to 8000 kDa for globular proteins, the GPC fractions are measured using a multi-element analysis method (eg ICP-MS, TXRF). Different forms of element bonding, which are distributed over different molecular weight ranges, are analyzed in the individual fractions. For example, there are 3 iron species with a molecular mass of 20, 50 and 70 kDa at the elution maximum and 2 copper species as well as other element bonding forms (multi-element species analysis).

To determine which of the detected element species are proteins, the cytosols are incubated with a proteinase and then separated using a GPC method. The fractions are again analyzed using ICP-MS (Inductively Coupled Plasma Mass Spectroscopy). It turned out that the smallest iron species is no longer visible in the chromatogram after protease treatment of the cytosol. This verifies that the 20 kDa iron bond form is a protein. The GPC fraction with the highest iron content of this iron species is further purified and isolated using the PNC-PAGE method according to the invention. An iron peak is shown in the electropherogram. The associated fraction with the highest iron content, which contains the isolated iron protein, can now be used for identification and structural analysis. MALDI-TOF-MS (atrix-assisted laser desorption i-onization - time of flight mass spectroscopy) is particularly suitable for this.

Since the genome of the investigated medicinal plant that produces this substance is known, the gene that codes for this protein can be precisely assigned using database searches.

Since iron in this case acts as a co-factor of an enzyme which is said to have a positive effect on metabolic processes in the human organism, it can now be produced in large quantities in an industrial-pharmaceutical manner because of its precise genetic identification made possible by the method according to the invention. For example, there is the possibility here of having biotechnological production of the enzyme carried out by microorganisms.

2nd example:

A zinc protein that elutes in the range of 180 kDa is also with the PNC-PAGE- according to the invention

Method further separated. It is not denatured at a pH of 10.00 and migrates during the electrophoretic separation from the anode. Zinc elutes in several fractions, with two zinc peaks visible at different locations in the electropherogram.

Structural studies showed that the zinc bond forms are substitution isomers with different isoelectric points, of which only one isomer is the medically effective one. To find out this difference, it is possible to determine the isoelectric points, the actual molecular mass and purity of the isolated zinc species by means of isoelectric focusing using protein standards.

Literature [1] K. Günther, G. Ji and B. Kastenholz, Fresenius J. Anal. Chem. (2000) 368: 281-287, "Characterization of high molecular weight cadmium species in contaminated vegetable food".

[2] Gang Ji, "Characterization of high molecular cadmium species in contaminated plant foods", Cuvillier Verlag Göttingen, 1997, pp. 1 - 113.

[3] BioRad, "Model 491 Prep Cell, Instruction Manual", pp. 1-47. [4] BioRad, "Acrylamide Polymerization - A Practical Approach", US / EG Bulletin 1156, pp. 1-8 (1989). [5] DPMA, "Container for the storage of samples at low temperatures", publication DE 197 25 768 A1, 1998, inventor: B. Kastenholz. [6] G. Weber and J. Messerschmidt, Fresenius J. Anal. Chem. (2000) 367: 356-358, "Total determination of metal-binding carbohydrates in plant extracts by using FIA with electrochemical detection". [7] G. Weber, J. Messerschmidt, A. von Bohlen and F. Alt, Fresenius J. Anal. Chem. (2001) 371: 921-926, "Effect of extraction pH on metal speciation in plant root extracts". [8] K. Guenther and B. Kastenholz, 'Handbook of Elemental Speciation', Rita Cornelis (Chief Editor ), Wiley, "Speciation of Thallium (Environment, Food, Clinical, Occupational Health)", submitted for review in 2002.

[9] K. Guenther and B. Kastenholz, 'Handbook of Elementary Speciation', Rita Cornelis (Chief Editor), Wiley, 'Speciation of Zinc (Environment, Food, Clinical, Occupational Health)', submitted for review in 2003 ,

[10] A. Wittershagen, P. Rostam-Khani, C. Rittmeyer, D. Bublak, G. Stahl, H. Rüterjans and BO Kolbesen, “Detection of metal cofactors in biological-organic systems. A comparison between ICP-OES and total reflection X-ray fluorescence analysis (TXRF) ", in, CANAS ^{^} 95 Colloquium Analytical Atomic Spectroscopy ", Bernhard Welz (ed.), Bodenseewerk Perkin-Elmer GmbH, 1996.

[11] R. Huber, Angewandte Chemie 101, 849-871 (1989).

[12] J. Deisenhofer, H. Michel, Angewandte Chemie 101, 872-892 (1989).

Claims

Patent claims

1. A method for isolating metal cofactors from biological-organic systems using preparative native continuous polyacrylamide gel electrophoresis (PNC-PAGE), characterized in that an acrylamide gel with a total monomer content of 4% (w / v) is used Electrophoresis buffer with a pH value of 10.00 is used, and 0.5 μl TEMED / ml gel and 5 μl APS / ml gel are used, the gel being polymerized out over a period of 69 hours.

2. The method according to claim 1, characterized in that in the electrophoretic separation glycerin is used with a volume fraction of 10% (v / v) based on the volume of the biological-organic system.

3. The method according to any one of claims 1 to 2, characterized in that the metal cofactors to be isolated are cleaned before use in the PNC-PAGE with a gel permeation chromatography (GPC).

4. The method according to any one of claims 1 to 3, characterized in that the acrylamide gel is coated with isopropanol for 60 min after casting and then rinsed with electrophoresis buffer.