RU2295510C1 - Method of preparing tritium-labeled humin and humin-like substances - Google Patents

Abstract

FIELD: labeled natural substances.

SUBSTANCE: invention provides a method for labeling humin and humin-like substances with tritium-containing agent, said agent being atomic tritium obtained via thermal catalytic dissociation of tritium on tungsten wire at pressure 0.3-0.6 Pa. Humin and humin-like substances are preliminarily deposited on reaction vessel walls utilizing lyophilization for aqueous solutions thereof. Thermal dissociation is effected by 5-10-sec pulses at tungsten wire temperature 1900-2000 K.

EFFECT: enabled uniform introduction of tritium and achieved radiation activity of preparation 69 to 254 GBc/g.

1 dwg, 2 tbl, 15 ex

Description

The invention relates to humic substances and can be used to study their physico-chemical properties, interaction with biological objects and distribution in the tissues of biological objects.

The introduction of a tritium label into the molecules of biologically active substances by atomic tritium bombardment of solid targets (MTAT) obtained by dissociation of tritium molecules on a tungsten wire was first proposed in [Shishkov AV, Filatov ES, Simonov E.F. . et al. // Dokl. USSR Academy of Sciences. 1976.V.228. S.1237-1241]. Using this method, it is possible to obtain labeled compounds of various classes, including biological macromolecules. By choosing the conditions for labeling, it is possible to ensure that the labeled compounds are obtained with acceptable specific radioactivity, and the side processes modifying the original compound are minimized. Using chromatographic methods for purification of the labeled product, the radiochemical purity of the obtained preparations was achieved, which meets modern requirements (≥97%). Labeled proteins, polysaccharides, and synthetic block copolymers obtained using MTAT were used to study the solubility of enzymes in nonpolar organic solvents [V.M. Mozhaev, K. G. Poltevsky, V. I. Slepnev, G. A. Badun, A. V. Levashov. Homogeneous solutions of hydrophilic enzymes in nonpolar organic solvents. New systems for fundamental studies and biocatalytic transformations. // FEBS. 1991. V.292, N 1-2. P.159-161], for the study of the immobilization of enzymes on chitosan and its derivatives [I.A. Veselova, D.L. Grigor'eva, T.N. Shekhovtsova, V.I. Korobkov, G.A. Badun. Using different polysaccharides and alkaline phosphatase immobilization. // Proceedings of Biocatalysis-2000: Fundamentals and Applications. Moscow. 2000 (June 10-15). P.173], to study the interaction of pluronics with lipid membranes of liposomes and blood cells [Melik-Nubarov N.S., Pomaz O.O., Dorodnych T.Y., Badun G.A., Ksenofontov A.L., Schemchukova O.B., Arzhakov S.A. Interaction of tumor and normal blood cells with ethylene oxide and propylene oxide block copolymers. // FEBS Lett. 1999. V.446. N.1. P.194-198], [Krylova O.O., Melik-Nubarov N.S., Badun G.A., Ksenofontov A.L., Menger F.M., Yaroslavov A.A. Pluronic L61 Accelerates Flip-Flop and Transbilayer Doxorubicin Permeation. // Chemistry. 2003. V.9, N16. P.3930-3936].

In addition, atomic tritium bombardment of biological macromolecules and their complexes is used to study their structure, and the exposure conditions are such that the structure and functional activity of complex biological entities is preserved [Shishkov AV, Goldanskii VI, Baratova LA, Fedorova NV, Ksenofontov AL, Zhirnov OP, Galkin av The in situ spatial arrangement of the influenza A virus matrix protein M1 assessed by tritium bombardment. // Proc. Natl. Acad. Sci. USA 1999. V.96. N.14. P.7827-7830], [Agafonov D.E., Kolb V.A., Spirin A.S. Proteins on ribosome surface: Measurements of protein exposure by hot tritium bombardment technique. // Proc. Natl. Acad. Sci. USA 1997. V. 94. N.24. P.12892-12897], [Baratova LA, Efimov AV, Dobrov EN, Feodorova NV, Hunt R., Badun GA, Ksenofontov AL, Torance L., Järvekülg L. In Situ Spatial Organization of Potato Virus A coat protein subunits assessed by tritium bombardment. // Journal of Virology, 2001. V. 75, No. 20, P. 9696-9702], [Dobrov E. N., Badun G. A., Lukashina E. V., Fedorova N. V., Ksenofontov A. L., Fedoseev V. M., Baratova L. A. Tritium planigraphy comparative structural study of tobacco mosaic virus and its mutant with altered host specificity. // Eur. J. Biochem. 2003. V.270. N16. P.3300-3308].

Comparison with other methods

The study of the properties of the components of humic substances (TB) and their behavior in various systems, including biological, is conveniently carried out using a radioactive label [Northcott, GL; Jones, C.S. Experimental approaches and analytical techniques for determining organic compound bound residues in soil and sediment. // Environ. Pollut., 2000, V. 108 (1), P.19-43]. In most cases, specific isotopically labeled chemicals are used that form various complexes or associates with HS [Kang KH, Dec J., Park H., Bollag JM Transformation of the fungicide cyprodinil by a laccase of Trametes villosa in the presence of phenolic mediators and humic acid. // Water Res 2002 Nov; 36 (19): 4907-4915], [Xu F., Bhandari A. Retention and extractability of phenol, cresol, and dichlorophenol exposed to two surface soils in the presence of horseradish peroxidase enzyme. // J Agric Food Chem 2003 Jan 1; 51 (1): 183-188], [Ertunc T., Hartlieb N., Berns A., Klein W., Schaeffer A. Investigations on the binding mechanism of the herbicide simazine to dissolved organic matter in leachates of compost. // Chemosphere 2002 Nov; 49 (6): 597-604], [Drzyzga O., Bruns-Nagel D., Gorontzy T., Blotevogel KH, von Low E. Anaerobic incorporation of the radiolabeled explosive TNT and metabolites into the organic soil matrix of contaminated soil after different treatment procedures. // Chemosphere 1999 Apr; 38 (9): 2081-2095]. Attempts have been made to introduce various radionuclides directly into the HS. In most cases, ¹⁴ C-labeled compounds were used for this and with their help the process of formation of humic like substances (GWPs) was studied [Dec J., Haider K., Bollag JM Release of substituents from phenolic compounds during oxidative coupling reactions. // Chemosphere 2003 Jul; 52 (3): 549-556], [Kappler, Andreas; Ji, Rong; Brune, Andreas. Synthesis and characterization of specifically 14C-labeled humic model compounds for feeding trials with soil-feeding termites. // Soil Biol. Biochem. 2000. V.32 (8-9), P.1271-1280], [Ji, Rong; Kappler, Andreas; Brune, Andreas. Transformation and mineralization of synthetic HC-labeled humic model compounds by soil-feeding termites. // Soil Biol. Biochem., 2000. V.32 (8-9), P.1281-1291]. Technetium-99m was also used to introduce the radioactive label [Rossler, D .; Franke, K .; Suss, R .; Becker, E .; Kupsch, H. Synthesis and chromatographic characterization of [Tc-99m] technetium-humic acid species. // Radiochim. Acta, 2000. V. 88 (2), P. 95-100], iodine-131, indium-111. The main problem that must be solved in this case is the choice of the radionuclide and the routes of its administration so that the physicochemical characteristics of the drugs do not change, and the distribution of the radionuclide in the "molecular ensembles" of the drugs adequately reflects the properties of the hepatitis B, and the labeled drugs must retain their properties for a fairly long time. It is also important that the procedure for introducing a radioactive label is universal, fast and relatively inexpensive. Based on the foregoing, it seems promising to use the radioactive isotope of hydrogen tritium as a labeling agent for the preparation of labeled HS. Hydrogen is an integral component of any organic molecule; its content in HS ranges from 3 to 6%. Tritium-labeled compounds due to the unique nuclear-chemical properties of tritium (half-life 12.3 years, maximum beta radiation energy 18.6 keV) are widely used in chemical and biochemical studies. There is a wide range of methods for introducing tritium into organic molecules [Isotopes in the Physical and Biochemical Sciences. V.1. Labelled Compounds. Edited by Buncel E. and Jones JR: Elsevier. NY. USA 1991], [Evans EA Tritium and its compounds. London Butterworths. 1966], [Moran, et al. Low pressure tritiation of molecules. United States Patent 4313911], [Zolotarev et al. Method for preparing biologically active organic compound labelled with hydrogen isotope. United States Patent 5026909]. However, most methods involve labeling in individual chemical compounds. An attempt was made to use catalytic isotope exchange to obtain tritium-labeled peat HBs, which were used to study their distribution in rat organs [Wang C., Wang Z., Yang C., Wang W., Peng A. The evidence for the incorporation of fulvic acid into the bone and cartilage of rats. // Sci. Total Environ. 1996. V.191. N 3. P.197-202]. However, this publication does not contain information on the characteristics of labeled preparations.

Closest to the claimed is a method of introducing tritium into humic and humic like substances by the interaction of these substances with a tritium-containing agent. Here, an attempt was made to introduce tritium into the HS of peat by reduction of -CHO groups using KB ₃ H ₄ [Pefferkorn E., Widmaier J., Elfarissi F. Aluminum ions at polyelectrolyte interface. II. Role in surface-area-exclusion chromatography of humic acid. // Colloid Polym. Sci. 2001. V.279. P.439-497]. In this case, the specific radioactivity of the labeled preparations turned out to be about 4 kBq / g, which is not a sufficiently high activity for many applications. The uniformity of tritium administration has also not been investigated.

The task was to create a method for introducing tritium into humic and humic like substances so that the uniformity of the distribution of the tritium label and its activity would be higher.

The problem was solved by the present invention.

In the method for producing tritium-labeled humic and humic-like substances by reacting humic and humic-like substances with a tritium-containing agent according to the invention, atomic tritium obtained by thermal catalytic dissociation of tritium on a tungsten wire at a pressure of 0.3-0.6 Pa is used before By interaction, humic and humic-like substances are applied to the walls of the reaction vessel by lyophilization of their aqueous solutions, and thermal dissociation is performed by pulses of 5-10 s at the temperature of the tungsten wire is 1900-2000 K.

Thus, a method for introducing tritium into humic and humic like substances is proposed, in which, when treated with atomic tritium obtained by thermal catalytic dissociation of tritium on tungsten at low (0.5 Pa) pressure, not individual chemical compounds are exposed, but finished preparations deposited on the walls of the reaction vessels by lyophilization of their aqueous solutions. The processing time is limited by short pulses (10 s), the temperature of the tungsten wire is limited to the range of 1900-2000 K. The selected conditions for the introduction of tritium provide a uniform substitution of hydrogen for tritium in any accessible positions of the treated molecules.

An important step in the preparation of the labeled preparation is the labile label removal stage, which is proposed in phosphate buffer with a concentration of 0.028 M at pH 6.8 using dialysis through membranes of the appropriate size (MWCO from 500 to 12000 Da) for at least 24 days at 4 ° C. Selection of the pore size of the membrane minimizes the loss of the drug during dialysis and maintains the molecular weight distribution.

At the final stage of preparation of the labeled sample, the distribution of the label by its components is analyzed using size exclusion chromatography. The uniformity of tritium label administration is controlled by comparing the yield profiles from the column of the starting and labeled preparations by UV absorption at 254 nm, as well as by the radioactivity profile of the labeled preparation.

It is proposed to use drugs obtained in this way in the study of their interaction with various biological objects.

EXAMPLES

Example 1. 1 ml of an aqueous solution of HA of coal with a concentration of 0.26 mg / ml was evenly distributed on the walls of the reaction vessel with a volume of 600 cm ³ and frozen with liquid nitrogen. Water was removed by vacuum lyophilization, collecting it in a trap with liquid nitrogen. The vessel with the target was attached to a special vacuum system, air was removed to a residual pressure of 0.001 Topp. Then the walls of the reaction vessel were cooled with liquid nitrogen and evacuation was continued to a residual pressure of 0.00005 Topp. Using a special palladium leak, a hydrogen-tritium mixture was introduced into the reaction vessel with a tritium content of 35% to a pressure of 0.004 Topp. He heated the tungsten wire located in the center of the reaction vessel to a temperature of 2000 K using alternating electric current. The heating time of the wire was 10 s. The residual gas was pumped out using a diffusion pump without cooling the reaction vessel with liquid nitrogen.

The tritium-treated coal HA target was dissolved in 2 ml of 0.028 M phosphate buffer pH 6.8. Its radioactivity was equal to 258 MBq. The solution was placed in a plastic tube and closed with a MWCO 2000 Da dialysis membrane. Dialysis purification of the drug was carried out for 28 days against 1 liter of phosphate buffer with its periodic replacement and measurement of radioactivity. The radioactivity of the labeled drug solution was 63 MBq.

To assess the identity of the starting and labeled preparation, purified from the labile label, they were gel-chromatographed fractionated on an Abimed chromatographic system, which included an HPLC pump, an autosampler, a glass column (⌀15 mm, L = 25 cm), and a UV spectrophotometric detector , fraction collector, ADC board for recording an analytical signal and a recording computer. The column was filled with Toyopearl TSK HW-55S gel (Toso-Haas, Japan) with a fractionation range of 1000-200000 Da for polydecountries. As the mobile phase, 0.028 M phosphate buffer with a pH of 6.8 was used. The volume of the eluted sample was 1 ml; the elution rate was 1 ml / min.

The UV absorption profiles of the starting and labeled preparations almost coincided (see drawing). Along with gel chromatographic analysis, it was fractionated by collecting 1.5 ml fractions. The radioactivity of the collected fractions was measured using liquid scintillation counting. The resulting profile for radioactivity coincided with the profile for UV absorption (see drawing). There were no radioactive products in the preparation with a retention time different from the original preparation. As a result of the analysis, it was found that the solution contained 0.25 mg of HA of coal. The specific radioactivity of the drug was equal to 254 GBq / g.

Example 2. The preparation of the preparation of HA coal for the introduction of tritium was carried out in the same way as in Example 1. Using a special leakage from palladium, a hydrogen-tritium mixture with a tritium content of 35% was added to a pressure of 0.004 Topp. He heated the tungsten wire located in the center of the reaction vessel to a temperature of 2000 K using alternating electric current. The heating time of the wire was 10 s. The residual gas was pumped out using a diffusion pump to a pressure of 0.0005 Topp, then a new portion of the hydrogen-tritium mixture was introduced. Repeated heating of the tungsten wire with an electric current to 2000 K for 10 s, after which the residual gas was pumped out without cooling the reaction vessel with liquid nitrogen.

The tritium-treated coal HA target was dissolved in 2 ml of 0.028 M phosphate buffer pH 6.8. Its radioactivity was equal to 444 MBq. The solution was placed in a plastic tube and closed with a MWCO 2000 Da dialysis membrane. Dialysis purification of the drug was carried out as in Example 1. The radioactivity of the solution of the labeled drug was 15.8 MBq at the end of dialysis purification.

Assessment of the identity of the original and labeled preparation using gel chromatographic analysis was performed in the same way as in Example 1. The profiles for UV absorption of the original and labeled preparations and the radioactivity profile of the labeled preparation were completely identical. As a result of the analysis, it was found that the solution contained 0.23 mg of HA of coal. The specific radioactivity of the drug was 69 GBq / g.

Example 3. The preparation of HA coal prepared for the introduction of tritium in the same way as in Examples 1 and 2. Hydrogen-tritium mixture with a tritium content of 35% was let in to a pressure of 0.004 Topp. The tungsten wire was heated to a temperature of 2000 K using alternating electric current. The heating time of the wire was 10 s. The residual gas was pumped out using a diffusion pump to a pressure of 0.0005 Topp, then a new portion of the hydrogen-tritium mixture was introduced. This procedure was repeated 4 times. The total heating time of the tungsten wire was 40 s.

The tritium-treated coal HA target was dissolved in 2 ml of 0.028 M phosphate buffer pH 6.8. Its radioactivity was equal to 924 MBq. The solution was placed in a plastic tube and closed with a MWCO 2000 Da dialysis membrane. Dialysis purification of the drug was carried out in the same way as in Examples 1 and 2. The radioactivity of the solution of the labeled drug was 44 MBq at the end of dialysis purification.

Assessment of the identity of the original and labeled drug using gel chromatographic analysis was done in the same way as in Examples 1 and 2. The profiles for UV absorption of the original and labeled drugs and the profile for radioactivity of the labeled drug completely coincided. As a result of the analysis, it was found that the solution contained 0.22 mg of HA of coal. The specific radioactivity of the drug was 200 GBq / g.

Example 4. 1 ml of an aqueous solution of HA of coal with a concentration of 0.3 mg / ml was evenly distributed on the walls of the reaction vessel with a volume of 400 cm ³ and frozen with liquid nitrogen. Further preparation of the drug for the introduction of the tritium label was carried out similarly to Example 1. Using a special leakage from palladium into the reaction vessel, a hydrogen-tritium mixture with a tritium content of 43% was added to a pressure of 0.004 Torr. He heated the tungsten wire located in the center of the reaction vessel to a temperature of 1880 K using alternating electric current. The heating time of the wire was 10 s. The residual gas was pumped out using a diffusion pump without cooling the reaction vessel with liquid nitrogen.

The target was dissolved in 1.5 ml of 0.028 M phosphate buffer pH 6.8. Its radioactivity was equal to 178 MBq. Labile tag removal was carried out over 25 days through a MWCO 2000 Da membrane. Chromatographic analysis was carried out similarly to the description in Example 1. The profiles for UV absorption of the starting and labeled preparations and the profile for radioactivity of the labeled preparation completely coincided. The specific radioactivity of the drug was equal to 153 GBq / g.

Examples 5-6. Charcoal HA preparations were prepared for the introduction of a tritium label similarly to Example 4. The tungsten wire was heated to 1750 and 1610 K for 10 s. The radioactivity of the solution after dissolution of the target was 60 and 39 MBq. After removing the labile tag for 25 days, a chromatographic analysis was carried out similarly to Example 1. The profiles for the UV absorption of the starting and labeled preparations and the profile for the radioactivity of the labeled preparation completely coincided. The specific radioactivity of the drugs was 42 and 17 GBq / g.

Examples 7-11. Targets of preparations of humic acids of peat PHA-Sk03, peat sulfonic acids PFA-Sk03, soil humic acids SHA-Ctl-00, soil sulfonic acids SFA-Ctl-00 and SFA-Pg-96 were prepared in reaction vessels with a volume of 600 cm ³ as described in Example 1. To introduce tritium, a hydrogen-tritium mixture was used at a pressure of 0.004 Topp. The heating time of the tungsten wire was 10 s, the temperature of the tungsten wire was maintained equal to 2000 K. Dissolution of the targets, dialysis purification and chromatographic analysis were performed as described previously. Data on the amount of the substance taken to prepare the targets, on the composition of the hydrogen-tritium mixture and the radioactivity of the preparations at different stages of purification are shown in Table 1. For all preparations, good agreement was obtained between the absorption profiles coming out of the column at 254 nm for the starting and labeled preparations, and also the radioactivity profile of the labeled drug.

Examples 12-13. Targets of preparations of the humic-like substance ligfola-16 (a product of the hydrolysis of wood lignin) were prepared by lyophilization of 1 ml of a solution with a concentration of 0.64 g / l on the walls of reaction vessels with a volume of 600 cm ³ and a tritium label was introduced as described in Example 1. To remove the label from labile positions used two approaches: the labeled preparation was kept for a long time at 4 ° C and then lyophilized; the labeled preparation was purified by dialysis at 4 ° C. as described in Example 1.

During gel chromatographic fractionation of the starting and labeled preparations, it was found that the elution curve of the labeled preparation purified by lyophilization is almost identical to the original preparation. Both in the initial preparation and in the labeled analogue, a pronounced shoulder is observed in the region of low molecular weights, which indicated the presence of a low molecular weight component in this preparation of ligfol. At the same time, the elution profile of the drug purified by dialysis is significantly different from the original. The maximum elution curve of the dialyzed preparation was shifted toward higher molecular weights. In this case, there was practically no shoulder in the region of low molecular weights. In addition, the peak area indicated a significant (33%) loss of the drug during dialysis. The indicated nature of the changes is explained by the fact that the low molecular weight compounds that make up the drug were removed during dialysis treatment. At the same time, for both labeled preparations, a good coincidence of their yield profiles for UV absorption and radioactivity was observed, indicating a uniform distribution of tritium over the components of the preparations. Table 2 summarizes the main characteristics of labeled ligfol preparations.

Example 14. Determination of the bioaccumulation factor of coal HA by bacteria. To determine the bioaccumulation factor of coal HA by bacteria, the tetracycline-resistant strain of gram-negative bacteria Escherichia coli was grown by deep cultivation at 37 ° C in M9 medium (Na ₂ HPO ₄ × 12H ₂ O 15.1 g / l, KH ₂ PO ₄ 3.0 g / l, NaCl 0.5 g / l, NH ₄ Cl 1.0 g / l, MgSO ₄ × 12H ₂ O 0.5 g / l, CaCl ₂ 5.6 mg / l, glucose 0.25 g / l) for 10-14 hours. The media were autoclaved at 120 ° С for 30 min. Sterilization of glucose solutions, calcium and magnesium salts and the preparation of coal HA was carried out by filtration through a membrane filter with a pore size of 20 μm; these components were added to the medium separately. Tetracycline at a concentration of 30 mg / l was used as a bacteriostatic agent, it was added to the medium immediately before inoculation. The pH of the medium was adjusted to 7.5 using NaOH.

An unlabeled preparation of coal HA in concentrations of 5-50 mg / l and a labeled preparation of coal HA were added to the nutrient medium to create a specific radioactivity of solutions of 1500-2000 Bq / ml. Then, the bacterial suspension was centrifuged at 5000 rpm for 30 minutes, and the equilibrium concentration of HA of the coal was determined in the supernatant. Next, the supernatant was replaced with an equivalent volume of M9 medium, the bacteria were resuspended, and the amount of HA of coal adsorbed by the bacteria was determined in the resulting suspension.

Bacterial growth was monitored by absorbance at 600 nm (OD600). First, for a series of suspensions with different bacterial contents, OD600 and wet weight of the bacteria were measured, which was determined by weighing the precipitate formed by centrifuging the bacterial suspension at 5000 rpm for 30 minutes. Then calculated the dependence of the fresh weight of bacteria from OD600:

Bacteria weight (g / l) = 2.5167 × OD600 + 3.2158.

Next, the weight of bacteria was calculated according to the obtained dependence.

Based on the obtained data, the adsorption isotherms of coal HA were constructed by bacteria and the bioaccumulation factor was calculated as the tangent of the angle of inclination of the isotherm. The resulting bioaccumulation factor was 3.2 l / kg.

Example 15. Determination of the amount of HA of coal received in bacterial cells. To determine the amount of HA of coal entering the bacterial cells, E. coli bacteria were expanded as described in Example 14. The bacteria were separated from the culture medium by centrifugation at 5000 rpm for 30 minutes. The supernatant was replaced with an equivalent volume of M9 medium, the bacterial sediment was resuspended, and the amount of HA of coal adsorbed by bacteria was determined by the specific radioactivity of the supernatant. Then, bacterial cells were lysed in suspension by the addition of chloroform. The suspension with disrupted cells was again centrifuged at 5000 rpm for 30 minutes, thus separating debris from the internal contents of the cells. Based on the specific radioactivity of the supernatant, the amount of HA of coal delivered to the cells was calculated. It turned out that 50% of the total amount of the drug absorbed by the cells enters the bacterial cells.

Claims (1)

A method for producing tritium-labeled humic and humic-like substances by the interaction of humic and humic-like substances with a tritium-containing agent, characterized in that atomic tritium obtained by thermal catalytic dissociation of tritium on a tungsten wire at a pressure of 0.3-0.6 Pa is used as a tritium-containing agent , before the interaction, humic and humic-like substances are applied to the walls of the reaction vessel by lyophilization of their aqueous solutions, and thermal dissociation is performed by pulses of 5-10 s at tungsten wire temperature 1900-2000 K.