EP0528009A1

EP0528009A1 - Method for removing species containing primary amino groups from solutions

Info

Publication number: EP0528009A1
Application number: EP19920907058
Authority: EP
Inventors: Andreas Zucker
Original assignee: Bio Rad Laboratories GmbH
Current assignee: Bio Rad Laboratories GmbH
Priority date: 1991-03-05
Filing date: 1992-03-05
Publication date: 1993-02-24
Also published as: DE4106984A1; EP0508106A3; WO1992015384A1; EP0508106A2

Abstract

Il est décrit un procédé de séparation, à partir de solutions, d'espèces contenant des groupes amino primaires, caractérisé en ce qu'on fait réagir l'espèce avec un support insoluble dans l'eau contenant des groupes HS et un dialdéhyde de formule (I), dans laquelle R1 et R2, qui peuvent être identiques ou différents, désignent un hydrogène ou un groupe hydrocarbure (groupe hydrocarbyle) éventuellement substitué, ou forment ensemble un noyau ou un système de noyau aliphatique, aromatique ou hétérocyclique éventuellement substitué, de manière à obtenir un complexe insoluble dans l'eau, répondant à la formule (II) dans laquelle Tr désigne le support insoluble dans l'eau, et Sp le reste de l'espèce contenant les groupes aminés primaires, et en ce qu'on élimine le complexe de la solution. Le procédé peut être utilisé, en tant qu'étape de séparation, pour le dosage d'aminoacides secondaires, telles que les hydroxyprolines, pour l'élimination de l'albumine dans les liquides du corps humain, pour la stérilisation de solutions aqueuses et pour le dosage spectroscopique indirect d'espèces contenant des groupes amino primaires.There is described a method of separation, from solutions, of species containing primary amino groups, characterized in that the species is reacted with a water-insoluble support containing HS groups and a dialdehyde of formula (I), in which R1 and R2, which may be identical or different, denote an optionally substituted hydrogen or a hydrocarbon group (hydrocarbyl group), or together form an optionally substituted aliphatic, aromatic or heterocyclic ring or ring system, of so as to obtain a water-insoluble complex, corresponding to formula (II) in which Tr denotes the water-insoluble support, and Sp the rest of the species containing the primary amino groups, and in that remove the complex from the solution. The method can be used, as a separation step, for the determination of secondary amino acids, such as hydroxyprolines, for the elimination of albumin in liquids from the human body, for the sterilization of aqueous solutions and for indirect spectroscopic determination of species containing primary amino groups.

Description

Process for the separation of species containing primary amino groups from solutions

DESCRIPTION

The invention relates to a method for separating primary amino-containing species from solutions and various applications of this method.

It is known to convert primary amines and amino acids and proteins with o-phthalaldehyde and mercaptoethanol to an isoindole derivative. According to Analytical Biochemistry 54, 102 to 114 (1973), this method is used to determine peptides and proteins after separation by gel electrophoresis using fluorescence analysis. The use of mercaptoethanol results in an increase in the fluorescence intensity. The quantitative separation of species containing primary amino groups is not intended or achieved by this process. The object of the invention is to provide a simple method for separating primary amino group-containing species from solutions, since these species frequently interfere with the determination of other species, for example secondary amino acids.

To achieve this object, it is proposed according to the invention that the species containing primary amino groups with a water-insoluble carrier containing HS groups and a diaidehyde of the formula

R

CHO

R

1 2 wherein R and R, which may be the same or different, represent hydrogen or an optionally substituted hydrocarbon group (hydrocarbyl group), or together form an optionally substituted aliphatic, aromatic or heterocyclic ring or a ring system, to form a water-insoluble complex of the formula

S-Tr

N Sp

! C

W ^ ^ CH

in which Tr represents the water-insoluble carrier and Sp represents the rest of the species containing primary amino groups, and removes the complex from the solution. The method according to the invention allows species containing primary amino groups to be removed from the solution to be investigated in one work step. This is achieved by a specific chemical reaction in which the species containing primary amino groups are covalently bound to the water-insoluble carrier containing the HS groups and are thus removed from the solution as a water-insoluble complex.

According to the method according to the invention, primary amines, primary amino acids, primary amino groups-containing peptides, proteins or carbohydrates or microorganisms (eg cells, bacteria or viruses) with primary surface amino groups can be removed from the solution as primary amino group-containing species be removed.

As water-insoluble carriers containing HS groups, polymeric organic compounds, e.g. polymeric carbohydrates are used which are obtainable by reacting a polymeric carbohydrate activated with an oxirane, such as agarose or sepharose, with hydrogen sulfide or an acidic sulfide. Such carriers have already been used as solid phases in affinity chromatography for the purification of proteins (cf. Methods in Enzymology, Vol. 182, 357 to 369 (1990)); however, the proteins are not covalently bound to the solid phase.

Furthermore, supports according to the invention based on a water-soluble inorganic substance can be used, which are obtainable by activating an inorganic substance containing a free OH group either (a) by treatment with an oxirane or siloxane and then with hydrogen sulfide or an acid sulfide or (b) a thiosilane. Preferred inorganic water-insoluble substances are silica gels or aluminum hydroxide gels. Such gels are known as intermediates in the synthesis of chiral support materials for chromatography. As dialdehydes, those can be used in which di

1 2

R and / or R are substituted by auxochromic or bathochromic groups. In this way, the adsorption spectrum of the complexes obtained can be shifted into the longer-wave range, so that the complexes can be analyzed spectroscopically.

Dialdehydes which are preferably used are those with aromatic rings, in particular o-phthalaldehyde or o-naphthodialdehyde

The method according to the invention can be used as a separator in the determination of secondary amino acids, such as hydroxyprolines. These methods interfere with primary amino acids, which is why their quantitative separation before carrying out the determination is desirable.

Hydroxyprolines are part of collagen. In the case of bone cancer, the connective tissue around the bones is destroyed, and the hydroxyprolines are found in the blood and urine. The method according to the invention can therefore be used to diagnose bone cancer. The same applies to the diagnosis of osteoporosis, where the connective tissue is also broken down. The method can also be used for the analysis of foods. For example, the collagen content of sausages can be determined.

The method according to the invention can also be used to de-proteinize (deproteinize) liquids, in particular body fluids, such as blood and urine, or also beverages, such as beer, wine and fruit juices. Protein substances are particularly troublesome when electrodes are used to determine the electrolyte, since the electrodes are smeared by the protein substances. Furthermore, the method according to the invention can be used for germ elimination and for removing antigens.

Another application of the method according to the invention is the determination of species containing primary amino groups in a solution. In this process, the procedure is followed in that the solution is reacted with an excess of a bathochromic or auxochromic substituent-containing dialdehyde and the HS-insoluble, water-insoluble carrier, the water-insoluble complex obtained is separated and the solution which is not bound in the complex is dissolved Proportion of the dialdehyde determined spectroscopically.

The invention is illustrated by the examples below.

example 1

Preparation of an agarose carrier containing HS groups

30 g agarose beads (6% degree of crosslinking) are washed with water and filtered. The filtrate is suspended in 24 ml of 1 M NaOH. 0.75 ml epichlorohydrin are added at room temperature with stirring. After stirring for 15 minutes, the mixture is stirred at 60 ° C. for a further 2 hours. The beads are then washed with water until the eluate has a neutral pH. It is then washed three times with 0.5 M sodium phosphate (pH 6.2). Immediately afterwards, the beads are filtered off and placed in 30 ml of 2 M sodium thiosulfate. After stirring for six hours at room temperature, 4 ml of diethothol (8 mg / ml) in 1 mM EDTA are added. After 30 minutes, the product is filtered off with suction. Then it is washed with different solutions; first with 300 ml 0.1 M NaHCO, 1 M NaCl, 1 mM EDTA,

3 then with 300 ml of 1 M EDTA and finally 300 ml of 10 mM

Sodium acetate pH 4, 1 mM EDTA. Example 2

Preparation of a silica gel carrier containing HS groups

100 g of untreated silica gel (precipitated silica) are mixed with 500 ml of toluene and stirred to achieve a homogeneous consistency. Then 50 g of trimethoxy-mercaptopropylsilane are added and the mixture is boiled under reflux for 4 hours. After cooling, the supernatant solution is decanted, the precipitate is washed on a suction filter with ethanol and sucked dry.

Example 3

Separation of catecholamines

200 μl of an aqueous solution of (1) adrenaline, (2) noradrenaline, (3 ⁾ N-methyldopamine and (4) dopamine (10 μg each) are placed on an HPLC column (100 × 4.6 mm, 3 μm) ) and at a temperature of 40 ° C and a flow rate of 1.5 ml / min with a mobile phase containing 32 vol. CH C, 0.1 vol-5. Trifluoroacetic acid and 68 vol-5 H 0, eluted and then detected in a UV spectrometer (254 n). According to the four components of the solution, four signals are visible.

200 μl of the same solution are mixed with 0.5 ml of a borate buffer ⁽ 0.5 M, pH = 8), 1 mg of o-phthalaldehyde and 300 mg of the agarose carrier from Example 1 containing HS groups. The mixture is shaken for 3 minutes and allowed to settle. 10 μl of the supernatant are applied to the HPLC column and, as indicated above, eluted with the mobile phase. The chromatogram only shows the signals of components (1) and (3), ie the secondary amines. The components ⁽ 2 ⁾ and (4), ie the primary amines, no longer appear in the chromatogram. The same result is obtained if the o-phthalaldehyde is replaced by naphthodialdehyde.

Example 4

Removal of primary amino acids from urine as a precursor for the determination of hydroxyproline

For the hydrolysis of the proteins, 200 μl urine are heated in a Pyrex glass with Teflon stopper together with an acidic cation exchanger at 150 ° C. for 1 hour. The amino acids are dissolved with 250 μl borate buffer (0.5 M, pH = 8). Ma lets the cation exchanger settle and adds 200 .mu.l of the supernatant with 20 .mu.l of an o-phthalaldehyde solution and 300 mg of the HS group-containing silica gel carrier from Example 2. The suspension obtained is shaken for 3 minutes and then derivatized with 100 .mu.l fluorenylmethyloxicarbonyl - added chloride (FMOC) solution (2 mg / ml CH CN). The derivatives

3 sation of the amino acids is used for the purpose of spectroscopic

Analysis ⁽ shift of the absorption maximum after longer

Wavelengths).

After a further 2 minutes in the vibrator, the suspension is centrifuged for 2 minutes at 13,000 rpm in an Eppendorf centrifuge.

10 ^{μl of} the supernatant are then applied to an HPLC column and eluted as in Example 3. Only the secondary amino acids are found in the chromatogram (detection with UV, 254 nm ⁾

For the quantitative determination of the trans-4-hydroxyproline, a known amount of an amino acid with secondary amino groups, for example N-methyl taurine, can run as an internal standard. Example 5

Determination of hydroxyproline in sausage containing collagen

About 4 g of a well homogenized liver sausage sample are weighed to the nearest 1 mg in a 100 ml round or Erlenmeyer flask. 30 ml of hydrochloric acid are added and the solution is heated to a gentle boil and boiled under reflux for 6 hours

The hydrolyzate is transferred quantitatively with water into a 500 ml volumetric flask, mixed with about 5 ml of petroleum spirit and filled up to the mark with water such that the petroleum gasoline layer with the fat dissolved therein is above the mark ¬ layer removed by suction and the aqueous phase filtered into a 500 ml Erlenmeyer flask.

A suitable dilution is prepared from the hydrolyzate so that the expected hydroxyproline concentration is in the range from 0.6 to 2.4 μg / ml. This is done by diluting 10 ml of the hydrolyzate with water in a 250 ml volumetric flask.

Using 200 μl of the hydrolyzate, the steps of removing the primary amino acids, the derivatization with FMOC and the spectroscopic determination of the hydroxyproline according to Example 4 are carried out.

Since hydroxyproline is an essential component of collagen, the collagen content of the sausage can be determined indirectly in this way. Example 6

Determination of electrolytes in the blood

200 .mu.l of blood are mixed with 500 .mu.l of borate buffer (0.5 M, pH = 8), 300 mg of the silica gel carrier from example 2 and 1 mg of o-phthalaldehyde containing HS groups. The suspension is shaken for 3 minutes and allowed to settle. The proteins contained in the blood are cova- via their primary NH groups

2 lent tied to the carrier. This can already be seen from the fact that the carrier is colored red by the erythrocytes.

The colorless supernatant is protein-free (biuret test negative).

+ + +,. The alkali cations (Li, Na, K) are in the supernatant

Determined using a conventional ion-sensitive electrode.

About twice as many determinations can be carried out as with untreated blood before the electrode becomes unusable.

Example 7

Sterilization of aqueous solutions

200 μl of rainwater are mixed with 1 mg of o-phthalaldehyde and 300 m of the silica gel carrier from Example 2 containing HS groups and shaken for 3 minutes. A sample of the supernatant is applied to an agar culture medium and incubated at 37 ° C. in the incubator for 2 days. No bacterial growth can be determined. An untreated sample of the rain water shows strong bacterial growth after 2 days of incubation. Example 8

Determination of a primary amino acid by difference analysis

A solution of 20 mg of o-naphtholdialdehyde in 1 ml of a 30 3 ml of acetonitrile solution (remainder of water) is mixed with 5 mg of alanine, dissolved in 1 ml of water, and with 200 mg of the silica gel carrier from Example 2 containing HS groups. The suspension is shaken for 3 minutes and centrifuged for 2 minutes. The extinction of the supernatant solution is measured in a photometer. By applying the Lambert-Beer law, the alanine content of the test solution can be calculated from the decrease in dialdehyde concentration.

Claims

_ 1 . - PATENT CLAIMS

1. A process for the separation of primary amino group-containing species from solutions, characterized in that. the species with a water-insoluble carrier containing HS groups and a dialdehyde of the formula

1 row

CHO

I.

CHO

2

R _'

S-Tr

1 R / - • C

l N - Sp

C / R / ^■ CH

in which Tr represents the water-insoluble carrier and Sp represents the rest of the species containing primary amino groups, and removes the complex from the solution. -. 2 -

2. The method according to claim 1, characterized in that the primary amino group-containing species is a primary amine, a primary amino acid, a primary amino group containing peptide, protein or carbohydrate or a microorganism with primary surface amino groups.

3. The method according to claim 1 or 2, characterized in that one uses a carrier based on polymeric carbohydrates, which is obtainable by reacting a polymeric carbohydrate activated with an oxirane, such as agarose or Sepharose, with hydrogen sulfide or an acidic sulfide.

4. The method according to claim 1 or 2, characterized in that one uses a carrier based on a water-insoluble in organic substance, which is obtainable by the fact that a free OH-containing inorganic substance either (a) by treatment with an oxirane or Siloxane activated and then reacted with hydrogen sulfide or an acidic sulfide, or (b) reacted with a thiosilane.

5. The method according to claim 1, 2 or 3, characterized in that a silica gel or an aluminum hydroxide gel is used as the inorganic water-insoluble substance containing free OH groups.

6. The method according to any one of claims 1 to 5, characterized ge indicates that one uses a dialdehyde in which

1 2 the radicals R and / or R by auxochrome or bathochrome

Groups are substituted.

7. The method according to any one of claims 1 to 6, characterized ge indicates that the dialdehyde used is o-phthalaldehyde

8. Application of the method according to any one of claims 1 to as a separation step in the determination of secondary amino acids, such as hydroxyprolines.

9. Application of the method according to one of claims 1 to deproteinization (deproteinization) of body fluids, such as blood and urine.

10. Application of the method according to one of claims 1 to the sanitization of aqueous solutions.

11. Use of the method according to any one of claims 1 to 7 for the determination of primary amino-containing species in a solution, characterized in that the solution containing an excess of a bathochromic or auxochromic substituent containing dialdehyde and the HS groups converts the water-insoluble carrier, separates the water-insoluble complex obtained and determines the dissolved portion of the dialdehyde not bound in the complex by spectroscopy.