JPH0121836B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention provides glycosphingolipids useful as pharmaceuticals,
In particular, it relates to a method for purifying gangliosides having sialic acid residues. In recent years, gangliosides have been found to be unique active molecules localized on the cell surface that are involved not only in neurological functions, but also in cancer, inflammation, immunity, viral infections, hormone receptors, etc. Furthermore, it exhibits strong acidity due to the presence of sialic acid in its molecule, and through the carboxyl group of sialic acid, it forms ionic bonds with other basic molecules, transfers alkali metals, or forms lactones under certain conditions. Furthermore, sialic acid is water-soluble due to the presence of carboxyl groups, but it is known that micelles are formed when dissolved in water due to the ceramide in the molecule, and the acidity is determined by the amount of sialic acid contained in one molecule. determined by the number of When they have the same number of sialic acids, positional isomers exist that differ in the bonding position of the sialic acid, and their acidity differs slightly. Due to the diversity of gangliosides mentioned above,
Purification of gangliosides has proven extremely difficult. In the present invention, the nomenclature proposed by Svennerholm was used. That is, for mono-, di-, tri-, and tetrasialic acid-containing gangliosides, M, D, T, and Q are added after the abbreviation G, and each positional isomer is distinguished by a number after the letter. Furthermore, in this specification, unless otherwise specified, a mixture of mono-, di-, tri- and tetrasialic acid-containing gangliosides will be referred to as gangliosides. Gangliosides are abundantly contained in the nervous tissue of mammals, and their content in other organs is low. For this reason, bovine brain gray matter is usually used as a raw material for obtaining gangliosides. As a method for extracting gangliosides, the chloroform-methanol (hereinafter abbreviated as CM) mixed solvent method is generally used.
(fresh tissue, etc.), an appropriate CM mixing ratio is selected. Other methods using tetrahydrofuran (EGTrams, CJLauter, Biochim
Biophys Acta, 60, 350 (1962)) is also known. Gangliosides obtained by the C-M extraction method or the tetrahydrofuran method are usually purified using a basic ion exchanger such as diethylaminoethyl (DEAE), taking advantage of the acidity of gangliosides.
It is carried out by chromatography using cellulose or DEAE. Furthermore, silicic acid column chromatography is used to isolate gangliosides with different sialic acid contents. However, gangliosides in nervous tissues are
Due to the content of valent ions, it binds strongly to proteins, and this ganglioside-binding protein has extremely similar physicochemical properties to gangliosides, so it is extremely difficult to separate the proteins contained in gangliosides using ion exchange chromatography. It is. For example, the protein content of gangliosides purified by a combination of C-M extraction method and DEAE-Sephadex column chromatography is 3 to 8%.
It was found to be within the range of When using gangliosides for medicine or functional research, it is desirable to remove and purify contaminating impurities, especially proteins, as much as possible, but gangliosides obtained by known methods contain 3 to 8% protein. , this removal method poses a problem. Methods for removing and purifying proteins from substances to be purified are broadly classified into methods 1) to 5), which are listed below. 1) is a method called a solubility method, which includes a salting-out method, an organic solvent method, a heavy metal method, an isoelectric focusing method, and a nonionic polymer method. 2) is a chromatography method that includes adsorption/elution method, ion exchange method, and two-phase partition method, and 3) is called a molecular sieve method, which includes gel filtration method, ultrafiltration method, and ultracentrifugation method. There is. In addition, there are 4) electrophoretic methods and 5) biological methods. However, whichever method is used, there are restrictions depending on the physicochemical or biological properties of the substance to be purified and the protein to be removed, and the scale of purification. Gangliosides are known to form micelles in aqueous solutions or polar solvents, and mixed proteins have been found to behave very similar to gangliosides in chromatography using DEAE-cellulose or DEAE-Sephadex. , 3), 4) and 5) were found to be unsuitable. Regarding the isolation of components with different sialic acid contents of gangliosides using the chromatography method in 2), the following documents 1) to 3) are known, but the purpose of this method is to isolate each component. However, no detailed study has been conducted on the removal and separation of mixed proteins. 1 Susumu Ando, Miyoko Isobe, Yoshitaka Nagai, Biochimica et Biophisica Acta, 424, 98 (1976) 2 Takashi Momoi, Susumu Ando, Yoshitaka Nagai, Biochimica et Biophisica Acta, 441, 488 (1976) 3 G. Tettamanti, F. Bonali , S. Marchesini, Y. Zambotti, Biochimica et Biophisica Acta, 296, 160 (1973) The proteins contained in gangliosides are not a single component, and their physicochemical properties are slightly different, but both It turned out to be a water-soluble protein. In Documents 1) to 3), ganglioside components are isolated by a concentration gradient method using a water-containing mixed solvent. However, these methods are not practical when the purpose is not to isolate ganglioside components, and removal of contaminating proteins was found to be insufficient. Therefore, as a result of various studies on methods for removing proteins mixed in gangliosides, we established the following method. That is, after the gangliosides are dissolved in a mixed solvent at an arbitrary mixing ratio, the mixture is passed through an adsorbent column, the gangliosides and proteins are adsorbed onto the column, and the mixture is eluted by changing the composition ratio of the mixed solvent in stages. Finally, elution with a polar solvent is performed, but this operation is performed to elute the protein that remains adsorbed on the column, and is not directly related to purification and does not need to be performed in particular. After performing the above operations, the protein remaining adsorbed on the column is further separated and purified by a concentration gradient method using an appropriate buffer solution. The adsorbent according to the present invention is preferably silicic acid, acid clay, activated carbon, etc., and any one or two of them are preferable.
You may use a combination of two or more species. Synthetic adsorbents such as Amberlite XAD resin can also be used. In order to adsorb gangliosides on a column, they are dissolved in a mixed solvent of a non-polar solvent such as chloroform or methylene chloride and a polar solvent such as methanol or ethanol, but the mixing ratio of the non-polar solvent and polar solvent can be adjusted arbitrarily. . However, preferably the mixing ratio is between 1:1 (V/V) and 3:1 (V/V).
is desirable. Elution separation of gangliosides and proteins adsorbed on the column is carried out by first eluting with a nonpolar solvent such as chloroform or methylene chloride, and then using a mixed solvent in which polar solvents such as methanol or ethanol are gradually increased in the above nonpolar solvent. Elute. Although this mixing ratio is not particularly limited, it is desirable to increase the polar solvent content in the mixed solvent by 10%.
Further, the amount of the mixed solvent required for elution may be 2 to 10 times the column capacity, but preferably 4 to 8 times the column capacity. Concentrate the fractions eluted stepwise with a mixed solvent of a non-polar solvent and a polar solvent, mix the desired fractions, and perform crystallization by an appropriate method, such as recrystallization or freeze-drying. As a result, it is possible to obtain purified gangliosides in which the component composition ratio of gangliosides is almost unchanged from before purification and the protein content is extremely low. The target fraction herein refers to a fraction in which the polar solvent content in the mixed solvent is 50 to 60%. The method of the present invention utilizes the physicochemical properties of gangliosides and mixed proteins that have been adsorbed on a column to be eluted in stages with a mixed solvent of non-polar and polar solvents. without changing the component composition ratio of
This is an extremely useful method for removing proteins. Examples of the present invention will be described below, but the present invention is not limited only to these examples. Example 1 18g of ganglioside obtained from bovine brain by a known method
(sialic acid content 29.9%, protein content 54%)
It was dissolved in 200 ml of M (3:1 V/V) mixture and adsorbed onto a silica gel column (100 mesh, 6 x 40 cm) which had been thoroughly washed with the same mixture in advance. Then 1) Chloroform 4, 2) CM mixture (3:1V/V)
1, 3) C-M mixture (3:2V/V) 2, 4)
Elution was carried out stepwise with CM mixture (1:1 V/V) 2, 5) CM mixture (2:3 V/V) 2, and 6) methanol 2. After concentrating each fraction, sialic acid and protein were quantified, and the results shown in Table 1 were obtained. Fractions 4) and 5) were mixed and lyophilized to obtain 15.3 g of purified ganglioside. Yield 85
%, sialic acid content 28.7%, protein content 0.37%. The composition of each component before and after purification is chloroform-methanol-0.2% after being applied on a silica gel plate.
After developing with a calcium chloride aqueous solution (55:45:10V/V) developing solvent, spray the resorcinol reagent,
After developing color at 120°C for 10 minutes, it was quantified using a chromato scanner. The results are shown in Table 2.

[Table] C: Chloroform, M: Methanol

[Table] Example 2 10g of ganglioside used in Example 1 was mixed with methylene chloride-methanol (hereinafter abbreviated as MC-M).
Silica gel-acid clay (1:1 V/V) dissolved in 100 ml of mixed solution and thoroughly washed with the same mixed solution (1:1 V/V)
1W/W) was adsorbed onto the column. Then 1) methylene chloride 2, 2) MC-M (3:1) mixture 1,
3) MC-M (3:2V/V) mixture 1, 4) MC
-M (1:1V/V) mixture 2, 5) MC-M (2:
Stepwise elution was performed with 3V/V) mixture 2 and 6) methanol 1, and after concentrating each fraction, sialic acid and protein were quantified, and the results shown in Table 3 were obtained. Fractions 4) and 5) were mixed and lyophilized to obtain 8.62 g of purified ganglioside. Yield 86.2%, sialic acid content 29.1%, protein content 0.42%.

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Claims (1)

[Claims] 1. Gangliosides containing proteins are adsorbed onto inorganic adsorbents such as silicic acid, acid clay, and activated carbon, or synthetic adsorbents such as Amberlite XAD-2 resin, and elution is performed by changing the solvent composition ratio in stages. A method for removing proteins, which comprises selectively eluating and purifying gangliosides from an adsorbent without changing the component composition ratio of gangliosides after elution and removal of proteins from an adsorbent.