JPH07243972A - Quantitative determination method for trace protein in lipid - Google Patents

Abstract

PURPOSE:To quantitatively determine the trace protein in lipid with high sensitivity by dissolving the lipid in an organic solvent, reacting it with an acid solution, removing the lipid, binding a coloring matter, and measuring the absorbance. CONSTITUTION:A trichloroacetic acid aqueous solution is added to and reacted with a sample solution dissolving egg yolk phosphatidylcholine which is a phosphorus lipid, in ethanol, for example, the mixture is homogenized in ethanol, it is sucked and filtered with the filter paper for a quantitative analysis, and protein is adsorbed on the filter paper. Hot ethanol, ethanol/ether mixed liquid, chloroform, and ether are combined and sucked, the filter paper is washed and dried, it is dyed with a dyeing liquid dissolving Coomassie brilliant blue G-250 in acetic acid, and the extra dyeing liquid stuck to the filter paper is removed. The filter paper is dried, then a fixed quantity of a pigment releasing agent is added to release the coloring matter, the concentration of the released coloring matter is measured as absorbance, and the protein concentration in the sample is calculated based on the calibration curve of the standard protein.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for quantifying a trace amount of a protein contained in a lipid derived from a natural product, particularly a phospholipid.

[0002]

2. Description of the Related Art Proteins derived from raw materials are often present in naturally occurring lipids, and when lipids are used for various purposes, proteins become a problem, and it is required to clarify their structure and content. ing. In particular, phospholipids and cholesterol have been vigorously promoted in recent years by pharmaceutical manufacturers as one of drug delivery systems to be applied to liposome preparations. Currently, intravenous injection is the main method of administration of liposome preparations. When administered by intravenous injection, if a protein other than the medicinal component is contained in the liposome preparation, it may be recognized as an antigen and the immune function may be activated to cause a problem. Therefore, it has been required to quantify the amount of protein in phospholipids and cholesterol used as a base for liposome preparations.

Methods for purifying phospholipids include solvent fractionation and column fractionation, which are intended to fractionate specific phospholipids from a phospholipid mixture. Most of the proteins contained in phospholipids are lipoproteins, and the fractionated phospholipid fraction contains lipoproteins containing similar phospholipids as constituents, and it is difficult to completely remove them.

As a protein quantification method, a generally known method, for example, a nitrogen quantification method such as the Kjeldahl method,
Burette method (Gornoll, AG, Journal of Biologica
Chemistry 177, 751 (1949)), Lowry method (Lowry, O.
H., Journal of Biological Chemistry 193, 265 (195
1)), the absorbance method for measuring the absorbance at a wavelength of 280 nm, the fluorescence method using a fluorescent substance such as fluoresamine (Bohlen, P.,
Stein, Archives of Biochemistry and Biophysics 15
5, 213 (1973)) and a dye, and a dye-binding method for measuring the absorbance (Bradford, MM Analytic).
al Biochemistry 72, 248 (1976)), etc., but all of them measure in an aqueous system and are difficult to apply to lipids, and their sensitivity is not sufficient for quantifying a trace amount of protein. Furthermore, in these methods, when phospholipids and the like coexist, they become interfering substances and cannot be accurately quantified. In addition, for the purpose of increasing the sensitivity of protein quantification and eliminating the influence of interfering substances, a microquantitative method using dyes (Bram
hall, S., Noack, N., Analytical Biochemistry 31, 1
46 (1969)) was found. In this method, a sample is first fixed on filter paper and then reacted with an acid solution, but in the fixed state, the reactivity is poor and a measurement error is likely to occur.
Furthermore, when the sample is a trace amount of protein in lipid, it is difficult to solubilize a large amount of lipid again and completely remove it, and if it remains slightly, it will bind to the dye and affect the measured value. As a final means, a method of performing amino acid analysis after hydrolysis is performed, but the operation is complicated and an expensive amino acid analyzer is required.
In addition, this method is only an analysis as an amino acid, and a quantitative analysis as an actually existing protein molecule cannot be performed.

[0005]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a method for quantifying a trace amount of protein in lipid with high sensitivity, which solves the above-mentioned conventional problems.

[0006]

The present inventors have found that the detection limit can be increased and a lipid-containing protein can be isolated and quantified by improving the method for quantifying a trace protein using a dye, and the present invention is completed. Came to. That is, the present invention, a lipid derived from a natural product, in particular, a phospholipid is dissolved in an organic solvent or a mixed solvent of organic solvent and water, and after reacting with an acid solution, the lipid is removed, and a dye is bound to absorb the absorbance Is a method for quantifying a trace amount of protein in lipids.

The lipids used in the present invention include water-insoluble lipids, especially phospholipids, cholesterol and fatty acids.
Egg yolk phosphatidylcholine (EYP)
C), egg yolk phosphatidyl ethanolamine (EYP
E), phosphatidylcholine (P with various acyl chain lengths)
C), phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and the like.

The lipid solution used as a sample is appropriately a solvent of about 1 to 100 parts by weight with respect to 1 part by weight of the lipid, and the solvent to be used is not particularly limited, but one in which each lipid is solubilized, particularly, In the case of EYPC, primary alcohols such as ethanol in which the phospholipids and lipoproteins are soluble are preferable, and in the case of PS, PE, PI and PG, chloroform / methanol / water or hexane / isopropanol / water are added to 10/7. It is preferable to use a solvent mixed in various ratios such as / 3.

The acid used in the acid solution includes hydrochloric acid, acetic acid, trichloroacetic acid and the like, and the solvent includes water or a solvent in which a lipid is dissolved, and is preferably an aqueous trichloroacetic acid solution. The concentration of the acid is not particularly limited, but a concentration of 3 to 15% by weight after addition to the lipid solution is preferable. The acid solution is preferably used after cooling to 0 to 5 ° C. Add 10 to 200 parts by weight of the acid solution to 1 part by weight of the lipid solution, and add 0 to
Keep at 5 ℃ for 5 ~ 30 minutes, then heat to 50 ~ 100 ℃ for about 3 minutes.
Hold for 0 minutes. Next, after cooling to 0 to 5 ° C. to sufficiently insolubilize the protein, it is separated from the lipid.

The separation of the insolubilized protein and the lipid may be carried out by centrifugation or the like, but it is preferably immobilized on a support. The most suitable support is a filter paper for quantitative analysis, and No. 42 (Whatmann), No. 5C (ADVANTEC TO
(YO) and the like, but it is also possible to use other membrane filters and the like. As a solvent for washing the protein immobilized on the support, a combination of solvents capable of dissolving the lipid in the measurement sample is suitable, but particularly when the sample is EYPC, the measured value is obtained when sphingomyelin which is a contaminant remains. A solvent that can completely remove sphingomyelin is preferable because it has a large effect on For example, warm ethanol, a mixed solution of ethanol: ether = 1: 1, chloroform, ether and the like are used in combination.

Suitable dyes to be used are xylene brilliant cyanine G, Coomassie brilliant blue G-250, naphthalene blue black and the like. Washing after dyeing,
For decolorization and absorbance measurement, use Bramhaul (Bramha
ll) et al., using a 7 wt.
After washing several times at 60 ° C to remove excess dyeing solution and drying, add decolorizing solution of methanol: water: concentrated ammonia water = 66: 34: 1 to adjust the concentration of liberated dye at a wavelength of 610 nm. The absorbance is measured and the protein concentration in the sample is calculated from the standard protein calibration curve.

[0012]

The protein contained in the lipid of the present invention can be quantified with high sensitivity by the method for quantifying a trace amount of protein in lipid.

[0013]

EXAMPLES The present invention will be specifically described below with reference to examples. Example 1 Egg yolk phosphatidylcholine 800 mg was dissolved in ethanol to make 2 ml, and 25 μl of a sample solution was collected, and 7.5% by weight at 3 ° C.
2 ml of an aqueous trichloroacetic acid solution was added, and the mixture was cooled at 3 ° C for 15 minutes. Then, after heating at 80 ° C. for 30 minutes, it was cooled at 3 ° C. for 15 minutes. After adding about 2 ml of ethanol to make it uniform,
Suction filtered using a 2.1 cm diameter filter paper (Whatmann 42),
The protein was adsorbed on the filter paper. The filter paper was then washed twice with 2 ml of warm ethanol (40-50 ° C.) with suction, once with 2 ml of ethanol / ether mixture (1: 1), twice with 2 ml of chloroform and twice with 2 ml of ether. After drying the filter paper, Coomassie Brilliant Blue G-250
Was dissolved in 7% by weight acetic acid (10 mg / ml), 2 to 10 ml
A fixed volume of was added and stained at 50 ° C for 15 minutes. After removing the dyeing solution, in order to remove the excess dyeing solution attached to the filter paper,
Washing was repeated with a 7 wt% acetic acid solution at -60 ° C until the bag became white. After drying the filter paper, use methanol / water /
A certain amount of decolorizing solution of concentrated ammonia water (66/34/1) was added to liberate the dye, and the wavelength of the solution was changed to 61 within 15 to 60 minutes.
The absorbance was measured at 0 nm. The measurement value obtained by performing the same operation using 25 μl of ethanol was used as a blank, and the value subtracted from the value obtained previously was used as the actual measurement value.

The amount of protein was calculated from the standard curve of standard measurement. 25 μl of bovine serum albumin (BSA) added to ethanol solution (200 mg / 500 μl) of dimyristoylphosphatidylcholine (DMPC) in an amount of 10 to 100 ppm was sampled as a standard sample, and the same measurement as above was performed to obtain a calibration curve. Good linearity was obtained for the calibration curve. The obtained value was converted into the concentration of protein contained in the phospholipid by the following calculation formula.

[0015]

[Equation 1]

Measurements were made on 3 lots of egg yolk phosphatidylcholine, and the results were 50.1, 25.6 and 50.5 ppm. In this way, it was possible to quantify trace proteins of 100 ppm or less in phospholipids with high sensitivity.

Example 2 An ethanol solution of egg yolk PC (400 mg / ml) was added with a final concentration of
The measurement was carried out using a sample to which the BSA aqueous solution was added so that the concentrations became 33, 84 and 167 ppm. The measurement conditions were the same as in Example 1, the amount of protein was calculated, and the recovery rate was calculated by the following formula. As a blank, a solution prepared by adding the same amount of water as the BSA aqueous solution added to the ethanol solution of egg yolk PC was used for the measurement.

[0018]

[Equation 2]

From the above measurement, an average recovery rate of 96.3% was obtained, and good linearity was obtained between the amount of BSA added and the measured absorbance (FIG. 1).

Comparative Example 1 With respect to 3 lots of egg yolk phosphatidylcholine as in Example 1, the amount of protein contained was determined by using the ethanol solution (400 mg / ml) as a sample and the method of Bramhole et al. Add 25 μl of the sample solution to a 2.1 cm filter paper (Whatmann 4
2) Spot it on the surface and dry it with a dryer at 3 ℃
2 ml of 7.5 wt% trichloroacetic acid in water for 15 minutes. After heating at 80 ° C. for 30 minutes, the acid solution was removed. Filter paper with ether / ethanol = 1/1 (volume ratio) 2 ml to 1
Wash once with 2 ml of ether. After the filter paper was dried, 2 ml of a dyeing solution in which Coomassie Brilliant Blue G-250 was dissolved in 7% acetic acid (10 mg / ml) was added, and dyeing was performed at 50 ° C for 15 minutes. After removing the dyeing solution, washing was repeated with a 7% by weight acetic acid solution at 50 to 60 ° C. until the bag became white. After the filter paper was dried, 2 ml of the decolorizing solution (same as above) was added, and the concentration of the released dye was calculated by measuring the absorbance at 610 nm. The measurement results for each of the three lots are 5170 and 46.
It was 80,4870ppm. Such a high measured value is because when the measurement sample contains phospholipids, a large amount of phospholipids remains and binds to the dye if the washing before staining is not sufficient.

Comparative Example 2 Using egg yolk phosphatidylcholine as a sample, the washing after adsorbing the protein on the filter paper was carried out by the same method as in Example 1, and the others were measured by the same method as in Comparative Example 1. . A sample solution was prepared by adding BSA to an ethanol solution (400 mg / ml) of egg yolk phosphatidylcholine at 30, 60, 90 and 120 ppm. The measurement result is 38,
The results were 71, 62, and 54 ppm, and the recovery rate of protein addition was about 40 to 130%, which was not an accurate result. The above was able to remove phospholipids other than proteins by washing with various organic solvents, but unreacted substances were removed together with phospholipids in the reaction with the acid solution, so the recovery rate of addition It is thought that the result was low. When treating a sample in the presence of a large amount of substances other than proteins, it is difficult to carry out the reaction uniformly in the immobilized state. It shows that close measurements can be obtained.

Table 1 shows the measurement results of Example 1 and Comparative Example 1.

[0023]

[Table 1]

[Brief description of drawings]

FIG. 1 is a graph showing the linearity of measured values with respect to the amount added in the experiment of adding and recovering BSA to egg yolk phosphatidylcholine in Example 2.

FIG. 2 is a graph showing measured values with respect to the amount of addition of a BSA addition recovery experiment to egg yolk phosphatidylcholine in Comparative Example 2.

Claims (1)

[Claims] 1. A method of dissolving a lipid in an organic solvent or a mixed solvent of an organic solvent and water, reacting with an acid solution, removing the lipid, binding a dye, and measuring the absorbance. A method for quantifying trace proteins in lipids.