FR2714906A1 - Biomolecule solubilizers consisting of non-detergent zwitterions and their uses. - Google Patents

Abstract

Agents for solubilising biomolecules, particularly proteins, are disclosed. The agents consist of zwitterions having formula (I) or (II), wherein X is N or P; B is a hydrocarbon chain having 1-6 carbon atoms; R<1>, R<2> and R<3> are hydrocarbon groups, where R<1> and R<2>, taken together with X, may form a heterocyclic ring; R, taken together with X, is an unsaturated heterocyclic ring; and Y is SO3<->, OSO3<-> or COO<->. Said agents are useful in methods for purifying, solubilising or assaying proteins, especially by electrophoresis or chromatography.

Description

Biomolecule solubilization agents consisting of

non-detergent zwitterions and their uses.

The present invention relates to novel agents for solubilizing biomolecules such as proteins, which can be used to solubilize or maintain in solution biomolecules during processes such as methods of purification, separation and analysis of biomolecules, for example by means of biomolecules.

electrophoresis or chromatography techniques.

When purifying or separating proteins, solubilization problems are frequently encountered. This is due to the fact that the purification is always carried out in the liquid phase, while the starting material is often in insoluble form because the proteins are associated with each other or with other compounds. However, to obtain a good purification yield, it is important to ensure good dissolution of the proteins and then to maintain them in solution despite variations in experimental conditions such as pH and ionic strength, which could lead to precipitation. proteins. The use of protein solubilizers is therefore necessary in the case where the proteins are present at the start in an insoluble form and in the cases where certain experimental conditions having an effect on their precipitation are varied. The solubilizers used so far to achieve this result are detergents, salts and urea, but they present

all disadvantages.

The use of detergents for this purpose is described, for example, by Navarrete et al in Biochimica and Biophysica Acta, 728, 1983, p. 403-408. The detergents mentioned by these authors such as sodium dodecylsufate, Triton X-100, octylglucosides and zwittergents are effective because for the most part they have a strong power of protein solubilization but this is generally accompanied by a strong denaturing effect due to the destruction of hydrophobic type bonds which lead to the aggregation of proteins between them but are also responsible for the conformation of proteins, necessary for their biological properties. In addition, in addition to their high price, detergents have the drawback of forming micelles which are often of a large diameter, which limits their use in cases where dialysis processes, gels and certain columns are used. chromatography. The salts can be used as protein solubilizers, when the aggregation of the latter is due to the formation of ionic-type bonds between the protein molecules. However, the use of salts is impossible in all cases where an increase in the conductivity of the solution is detrimental, for example in electrophoretic processes and chromatography processes.

by ion exchange.

Urea can also be used as an agent for solubilizing proteins because it has a strong chaotropic power, and ensures the breaking of hydrogen-type bonds and of those involving dipoles. However, urea has two major drawbacks which are respectively its denaturing effect, and the risks of carbamylation of the free amines of the proteins by the cyanate groups formed.

by the spontaneous decomposition of urea.

Also, research was continued to find better biomolecule solubilization agents, exhibiting in particular the following properties: - do not cause denaturation of the biomolecules by modifying their conformation, - be able to be easily removed at the end of the operation to allow obtaining pure biomolecules and their lyophilization, - not to form micelles which are difficult to remove, not to interfere with the ionic strength of the solutions to which they are added, and

- be easy to manufacture and inexpensive.

The present invention specifically relates to novel solubilizing agents for biomolecules such as proteins, which exhibit

these advantageous properties.

According to the invention, the biomolecule solubilizer is constituted by a non-detergent zwitterion corresponding to one of the following formulas: Rl

RZ t + Y- (I) or R X + y-

R2-- ± --BBY

in which: - X represents N or P, -B represents a saturated hydrocarbon chain of 1 to 6 carbon atoms optionally comprising in its chain one or more oxygen and / or sulfur atoms, which may additionally contain one or more substituents consisting of OH groups, - R1, R2 and R, which may be identical or different, represent hydrocarbon, aliphatic, cyclic or aromatic groups, having in addition 7 carbon atoms, including or not comprising one or more -O- groups, -S- and / or -OH, R1 and R2 can also together form a saturated hydrocarbon chain of 4 to 8 carbon atoms, optionally comprising one or more OH substitutes and / or one or more oxygen and / or sulfur atoms , - R represents with X an unsaturated heterocycle with one or more rings containing at most 9 carbon atoms and one or more heteroatoms chosen from O, S, N and P, and optionally comprising one or more OH substituents, and

- Y represents SO3-, OSO3- or COO-.

The zwitterions used in the invention are different from the detergent zwitterions described by Navarrete et al in Biochimica and Biophysica Acta, 728, 1983, p. 403-408. Indeed, unlike detergent zwitterions which comprise alkyl groups having at least 8 carbon atoms, the non-detergent zwitterions of the invention do not have long hydrophobic chains; therefore, they have a very weak denaturing effect and they do not form micelles which are difficult to remove. Moreover, they can be easily removed at the end of the operation to give pure biomolecules and they do not interfere with the ionic strength of the solutions. Finally, they

are easy to synthesize and inexpensive.

Certain non-detergent zwitterions of the same type as those of the invention have been described in EP-A-0494686 However, in this case, they are not used to promote the solubilization of biomolecules, but play the role of surface modifying agents. in a process of separation by electrophoresis in capillaries, in order to avoid interactions between the charged internal surface of the capillaries and the biomolecules to be separated. Zwitterions derived from methacrylamide have also been used to make electrophoresis gels bearing sulfobetaine groups, intended for the separation of proteins, as described by Hjelmeland et al in Electrophoresis 1981, 2, p. 82-90. In this case, on the one hand, it is not a question of zwitterions of the same type as those of the invention, and on the other hand, these do not have the function of

solubilize biomolecules.

According to the invention, in the aforementioned formulas I and II, B may represent a saturated hydrocarbon chain having from 1 to 6 carbon atoms, optionally comprising in its chain one or more oxygen and / or sulfur atoms and optionally one or more substituents consisting of

by OH groups.

As examples of such hydrocarbon chains, mention may be made of the groups corresponding to the following formulas: - (CH2) 1- with 1 <1 <6, - (CH2) m-0- (CH2) n- with 1 <m <5, 1 <n <5 and 2 <m + n <6, (CH2) mS- (CH2) n, or - (CH2) m-CHOH- (CH2) n-, with m and n having the

meanings given above.

Preferably, B represents the group

- (CH2) 1- with 1 being equal to 3 or 4.

In the above formulas I and II, the aliphatic, cyclic or aromatic hydrocarbon groups used for R1, R2 and R3 can in particular be alkyl alkoxyalkyl, alkylthioalkyl, linear or branched with 1 to 7 carbon atoms, cycloalkyl groups of 5 with 7 carbon atoms and aryl groups with 5 to 7 carbon atoms. All these groups can also include a

or more OH substituents.

As examples of such groups, mention may be made of methyl, ethyl, propyl, butyl,

hydroxyethyl, cyclohexyl and phenyl.

According to the invention, when R1 and R2 together form a saturated hydrocarbon chain optionally comprising one or more OH substituents and / or one or more oxygen and / or sulfur atoms in the chain, R1 and R2 together with X form a heterocycle which can include other heteroatoms. As examples of such heterocycles, mention may be made of the piperidine, piperazine and morpholine nuclei. In this case, R3 is preferably an alkyl group, for example a methyl group. In the case of the zwitterion of formula II, o R forms with X an unsaturated heterocycle with one or more rings, it can be, for example, the pyridine, oxazole, thiazole, benzothiazole or benzoxazole ring. The non-detergent zwitterions used in the invention can be prepared by conventional methods, from sultones, lactones, (-chloroacids or corresponding chloroalcohols by reaction with amines or phosphines of formula: R2 or X R

3

In the case where Y represents S03-, the corresponding sultone is usually reacted with the amine or the phosphine, which corresponds to one of the following schemes:

> SO2 X 1R2R2- X -B - S03 -

R3R R3

0 S2 + X R OR X + - B - S03 -

in which B, X, R, Ri, R2 and R3 have the

meanings given above.

In the case where Y represents COO-, it is possible to react an m-chloroacid, a lactone or the acid

acrylic on the corresponding amine or phosphine.

In the case where Y represents OS03-, the zwitterion is usually obtained by carrying out the following steps: 1. reaction of a chloroalcohol of formula ClBOH with the amine or phosphine of formula: Ri X R2 or X R and

3

2.reaction of the product obtained in the first step

with chlorosulfonic acid HSO3C1.

The biomolecule solubilizers of the invention can be used in various methods involving biomolecules such as the methods of solubilization, extraction and purification of biomolecules, and the methods of separation of biomolecules by electrophoresis techniques or chromatography, for example separations by isoelectrofocusing, chromatofocusing and by ion exchange chromatography. These solubilizing agents can be used alone or as a mixture, and optionally in combination with other known solubilizing agents such as weakly denaturing detergents, for example the product marketed under the name TRITON X 100 or (tert-octylphenyl- (oligoethylene

oxide), octyl glucopyranoside and 3 [3-

cholamidopropyl] dimethylamonio propane sulfonate. The solubilizing agents of the invention can also be used for the renaturation of certain biomolecules such as enzymes, after these have been denatured, for example by a strongly denaturing solubilizing agent When these solubilizing agents are used in processes separation or purification of biomolecules by electrophoresis, they can be added to the solution of biomolecules to

to separate or to be purified, and / or to the separation medium.

Thus, in the case of gel electrophoresis methods, they can be added to the preparation solution and / or to the rehydration solution of the gel. In the case of liquid vein electrophoresis processes, we

can add them to conductive solutions.

In the case where these solubilizing agents are used in separation or purification processes by chromatography, they can be added to the solution of biomolecules to be separated or purified.

and / or conditioning and / or elution solutions.

In the case where these solubilizing agents are used for the renaturation of denatured biomolecules, they can be added to the solutions used.

in the process of renaturation.

The biomolecules to which the solubilizing agents of the invention apply can consist of any biological molecule, for example nucleic acids, carbohydrates,

lipids, coenzymes and in particular proteins.

The proteins can consist in particular of enzymes, plasma proteins, nuclear proteins, membrane proteins,

antibodies and structural proteins.

Other characteristics and advantages of the invention will appear better on reading the following examples, given of course by way of illustration and not limiting with reference to FIGS. 1.

to 10 appended.

Figure 1 is a diagram showing the efficiency of microsomal protein extraction

obtained in Examples 9 to 15.

Figure 2 is a diagram showing the maintenance yields of nuclear proteins in solution in dialysis solutions after

saline extraction.

FIG. 3 is a photograph illustrating the results obtained during one-dimensional gel electrophoresis (isoelectrofocusing) of a protein in the presence of a solubilizing agent of the invention. FIGS. 4 and 5 are photographs illustrating the results obtained during the separation by two-dimensional gel electrophoresis of a complex mixture of proteins in the presence and in the absence of a solubilizing agent in accordance with the invention. FIG. 6 is a diagram illustrating the enzymatic activity of O-galactosidase in the presence of various solubilizing agents in accordance with the invention. FIG. 7 is a diagram illustrating the enzymatic activity of 3-galactosidase as a function of the concentration of compound n 2 of the medium

dilution.

FIG. 8 is a diagram illustrating the percentage of enzymatic activity recovered in the presence of compound n 2 after denaturation of the

-galactosidase by urea.

FIG. 9 is a photograph illustrating the results of analysis by gel electrophoresis of the fractions obtained from a chromatography column in

presence and absence of compound n 2.

Example 1: Trimethylammonio methane carboxylate

(compound # 1).

This compound was purchased from Sigma, its

formula is given in Table 1 attached.

Example 2: Preparation of pyridinium propane sulfonate

(compound # 2).

The commercial product purchased from Fluka was then purified by passing it through an aqueous solution on

mixed ion exchange resins.

We thus obtain the compound n 2 responding

with the formula given in Table 1 attached.

Examples 3 to 8.

In these examples, 0.1 mole of propanesultone with butane sultone is reacted with 1 mole of the appropriate amine in 100 ml of an ethanol-water mixture (50/50 by vol). After 18 h of reaction at room temperature, the product obtained is purified by triple solubilization in water and crystallization in acetone. We thus obtain compounds n 3 to 8

responding to the formulas given in the attached table.

Examples 9 to 15: solubilization of microsomal proteins. In these examples, we use different

compounds for solubilizing microsomal proteins.

P3-X63-Ag8 mouse secreting plasmacytoma cells were cultured and the proteins labeled by incorporation of sulfur-labeled amino acids 35. The cells were recovered, washed in saline and lysed by lysis. osmotic for 30min, at 0 C in the following solution: - hydroxyethyl piperazine ethanesulfonic acid (HEPES): at 10mmol / l, - sodium hydroxide (NaOH): 5mmol / l, dithiothreitol (DTT): 2mmol / l, - chloride magnesium: 1, Smmol / l, and phenylmethyl sulfonyl fluoride (PMSF): 0, Smmol / l. Cells and cell debris are removed by centrifugation (1000g) for 5min. The supernatant is recovered, it is divided into aliquots and the microsomes are prepared by centrifugation (150,000 g) for 1 h. The microsome pellets are extracted (750 to 800 gg per pellet) using in each example 0.5Sml of an extraction solution whose composition is

given in table 2.

With the exception of Example 9, where the extraction is carried out at 20 ° C., the extraction is carried out in all cases for 1 h at 0 ° C., then the non-solubilized proteins are removed by centrifugation (200 000 g, 1 h). We collect the liquids

supernatants which contain the solubilized proteins.

The extraction yield is calculated by comparing the amounts of proteins solubilized by the solutions of Examples 10 to 15 to that solubilized by the solution of Example 9 taken as an extraction reference, because it contains a very effective solubilizing agent but strongly denaturing,

sodium dodecyl sulfate (SDS).

The extraction yields obtained, expressed as percentages of the quantity of proteins solubilized in Example 9, are presented in the

attached figure 1.

In view of this figure, it can be seen that the addition of compound n 2 to Triton X 100 makes it possible to achieve an extraction yield practically equal to that obtained with sodium dodecylsulphate; in the case of octylglucopyranoside (Octyl G), the addition of compound n 2 makes it possible to significantly increase the extraction yield but the results remain inferior to those obtained with

sodium dodecyl sulphate.

In the case of compound 3, a synergistic effect is also obtained with octylglucopyranoside, but this is not the case.

with the Triton X100.

It is therefore possible to obtain with the compounds of the invention protein extraction yields equivalent to those which are obtained with a very effective but highly denaturing protein solubilizer such as sodium dodecyl sulphate. Examples 16 to 18: keeping proteins in solution

nuclear after saline extraction.

In these examples, different solubilizers are used to maintain proteins

nuclear in solution.

BASC 6-C2 murine lymphoma cells are cultured and their proteins are labeled as above by incorporation of amino acids with 35S sulfur. After washing in saline solution, the cells are lysed (20min, OC) with 20 volumes of the solution having the following composition: - HEPES: 10mmol / 1, - NaOH: 5mmol / l, - DTT: 2mmol / 1, - spermidine : 0.75mmol / l, - spermine: 0.15mmol / l, - ethylenediaminetetraacetic acid (EDTA): lmmol / l, - triton X100: 1.25ml / l, - PMSF: 0.5mmol / l, and

- sucrose: 116g / l.

The nuclei are collected by low speed centrifugation (1000 g, 5 min) and washed by resuspension in the solution having the composition given above and recentrifugation. This operation is repeated twice. The nuclear proteins are then extracted for 1 h at 0 C with the following solution: - HEPES: 10mmol / l, - NaOH: 5mmol / l, - DTT: 2mmol / 1, EDTA: lmmol / l, - KCl: 0.8mol / l, and

- PMSF: 0.5mmol / l.

The nucleic acids and the non-solubilized proteins are removed after extraction, by centrifugation (200,000 g, 1 h). The solubilized proteins are then dialyzed against the solutions having the compositions given in Table 3, using in each case a volume of solution equal to 100 times.

the volume of the solubilized protein extract.

The yield of protein maintenance in solution is determined by comparing the quantity of protein in solution obtained after dialysis with that

obtained before dialysis.

The results obtained, expressed in% of the quantity of proteins solubilized before dialysis, are

given in Figure 2 attached.

In view of this figure, it can be seen that the yield for maintaining proteins in solution is much greater when the dialysis solution contains compound n 2 or compound n 3 (Examples 17 and 18). Examples 19 to 21: maintenance of proteins in solution

nuclear after saline extraction.

In these examples, the same procedure is followed as in Examples 16 to 18, but a solution having the following composition is used for the extraction of the nuclear proteins: - HEPES O: 10mmol / 1, - NaOH: 5mmol / l, - DTT: 2mmol / l, - EDTA: lmmol / l, - KC1: 0.8mol / 1, - PMSF: 0.5mmol / 1, and - 3 [3-cholamidopropyl] dimethylammonio propane sulfonate

(CHAPS): 5g / l.

After removal of the non-solubilized proteins and nucleic acids, by centrifugation under the same conditions as those described in Examples 16 to 18, the solubilized proteins are dialyzed against the solutions having the compositions given in Table 3 using in each case a volume of solution corresponding to 100 times the volume of extract. The results obtained, expressed as in the case of Examples 16 to 18 in% of the amount of proteins solubilized before dialysis, are given on

figure 2.

In view of this figure, it can be seen that the addition of compound n 2 or of compound n 3 to the dialysis solution makes it possible to greatly increase the

maintenance yield of proteins in solution.

Example 22: Separation of a protein by

gel isoelectrofocusing.

In iscelectrofocusing separations, proteins are brought to their isoelectric point by the action of a field

electric on a pH gradient.

Isoelectric focusing presents a formidable problem of solubility because proteins are brought to their isoelectric point where their solubility is minimal, in the absence of salts, which further contributes to increasing precipitation. Usually, isoelectrofocusing is carried out in the presence of 8mol / 1

urea (strongly denaturing condition).

In this example, instead of urea, a protein solubilizer in accordance with

to the invention consisting of compound n 2.

The isoelectrophoretic separation is carried out on a pH gradient using immobilized gels (Immobilins II from Pharmacia Uppsala Sweden) and prepared in the laboratory according to the

supplier recommendations.

The gels are washed, dried in the presence of

1.5% glycerol, and cut into 4mm wide strips.

They are then rehydrated in distilled water

with or without 1 mol / l of compound 2 of Example 2.

The protein sample to be separated which, in this case, consists of a fragment protein of kDa of fibronectin, is loaded on the surface of the gel and the focusing is carried out with a field of 3V / cm for 3h, 10V / cm for 3h, 100V / cm for

18h and 200V / cm for lh.

In this example, a one-dimensional gel is used which is stained with Coomassie blue as described by Neuhoff et al in Electrophoresis,

1988, 9, p. 255-262.

To perform Coomassie blue staining, fixation is carried out for 1 h in 2% H3PO4, 40% ethanol, followed by three rinses of min in 2% H3PO4, incubation for 1 h in (NH4) 2SO4 at 15 % and add Coomassie blue G 250

powder in this bath.

FIG. 3 shows the migration tracks of the protein 110 kDa fragment of fibronectin in the absence and in the presence of compound n ′ 2 in a linear gradient ranging from a pH of 7 to a pH of 4. On the top track, which corresponds to the migration of the protein in the absence of compound n 2, precipitation of the protein is observed before it reaches its isoelectric point, which results in a trail. On the other hand, on the bottom track which corresponds to the migration of the protein in the presence of 1 mol / l of compound n 2, we see that the protein migrates to its isoelectric point and we observe the separation of the isoforms into clear bands. , which shows that the protein remains soluble under these conditions.

Example 23: Separation of plasma proteins.

The same procedure is followed as in Example 22 to separate plasma proteins by isoelectrofocusing, either on a linear gradient from pH 3.5 to pH 5, with or without adding 1 mol / 1 gels to the rehydration solution of compound n 2. In all cases, two-dimensional gels are used, and the migration is followed by incubation of the bands for 10 min in a solution having the following composition: - urea: 6 mol / l, - glycerol: 30% , - Bis-tris: 0, 2mol / l, - HCl: 0, lmol / l, - sodium dodecyl sulfate: 2%, and - dithiothreitol: 1%, then for 10min in a 6mol / 1 solution of urea , 30% of glycerol, 0.2mol / 1 of Bis-tris, 0.1mol / l of HCl, 2% of dodecylsulphate of

sodium and 4% iodoacetamide.

The transfer onto polyacrylamide gel is then carried out by sealing the isoelectrofocusing gel with 1% agarose in a 0.2mol / 1 solution of Bis-tris, 0.1mol / 1 of HCl, 0.2% of sodium dodecyl sulfate and 0.002% bromophenol blue. The proteins are separated by electrophoresis (40 mA by gel of 160 mm X 200 mm X 1.5 mmn) then stained with Coomassie blue as in

example 22.

FIGS. 4 and 5 illustrate the results obtained during the migration from pH5 to pH3.5, in the absence of compound n 2 (FIG. 4) and in the presence of

Compound # 2 (Figure 5).

In FIG. 5, it can be seen that the migration from pH 5 to pH 3.5 leads to the appearance of well-defined spots, whereas in the absence of compound n 2, (FIG. 4) the focusing in the first dimension is weak, very few spots are observed and a large

number of trails.

Thus, in the presence of compound n 2, there is a reduction in the number of streaks, this proves that the insoluble proteins are made soluble by the

compound # 2.

Example 24: Conservation of the enzymatic activity of

0-galactosidase.

In this example, it is verified that the solubilizing agents of the invention have no denaturing effect on O-galactosidase since the latter retains its enzymatic activity when it is in

presence of the solubilizing agents of the invention.

For this purpose, we use a commercial suspension (760 units per ml) of 3-galactosidase which is diluted 250 times in a buffer: HEPES-NaOH (pH 7.8) 50mmol / 1, NaCl 200mmol / 1, DTT lmmol / l, Mg C12 lmmol / l containing lmol / l of compound 1 of Example 1 as solubilizing agent. The solution is left to incubate for 15 h at 8 C, then diluted 25 times, then its enzymatic activity is measured at 37 C,

by detection of hydrolysis of o-nitrophenyl 3-D-

galactopyranoside (ONPG) (2mmol / 1), which results in

an increase in optical density to 405nm.

For comparison, the same measurements are carried out 1) using a buffer not containing the solubilizing agent of the invention (control 1) and 2) by determining the enzymatic activity of a commercial suspension of dilute 3-galactosidase.

directly without incubation (control 2).

The results obtained, ie the enzymatic activity expressed in U / mg of enzyme are given in FIG. 6. In this figure, it is noted that the addition of compound n 1 (example 24) only decreases.

very weak enzymatic activity of beta

galactosidase since it is close to that of the control

n 1 without additive and of control n 2, without incubation. Examples 25 to 27: Preservation of activity

5-galactosidase enzyme.

In these examples, the same procedure is followed as in Example 24 to study the enzymatic activity of 3-galactosidase, but using as solubilizing agent lmol / l of compound n 5 (example 25), lmol / l of compound n 2

(example 26), and lmol / l of compound 4 (example 27).

The results obtained are also given

in figure 6.

In this figure, it can be seen that compounds 2 and 5 (examples 26 and 25) have no denaturing effect but, on the contrary, have an effect

beneficial on the enzymatic activity of the -

galactosidase. Example 28: Effects of the Concentrations of Compound No. 2

on the activity of 0-galactosidase.

In this example, we study the effect of increasing concentration of compound n 2 on

the activity of 0-galactosidase.

For this study, a commercial suspension of 0-galactosidase (760 units / ml) is diluted 250 times in a buffer: HEPES-NaOH (ph 7.8) 50 mmol / l, NaCl mmol / l, DTT 1 mmol / l, MgC12 1 mmol / l. We incubate

each solution for 15 h at 8 C.

The solutions are then diluted 25 times,

then the enzymatic activity of the P-

galactosidase as in Example 24, in the presence of

concentrations of compound n 2 ranging from 0 to lmol / l.

The results obtained are represented on

Figure 7 which illustrates the activity of the 0-

galactosidase (en.mol of hydrolyzed substrate (ONPG) per mg of enzyme) as a function of the concentration of compound n 2 (in mol / l) of the medium for determining

enzymatic activity.

These results show the little influence of high concentrations of compound n 2 on the activity

of the enzyme.

Example 29: Effects of the Concentrations of Compound No. 2

on the activity of alkaline phosphatase.

In this example, the effect of increasing concentrations of compound n 2 on

alkaline phosphatase activity.

For this study, a commercial suspension of alkaline phosphatase is diluted 250 times in a tris-HCl buffer (pH 8.8) 50mmol / 1, NaCl 200mmol / 1, ZnCl2 0, lmmol / l, MgCl2 0, Smmol / l containing 0 , 0.2 or lmol / l of compound n 2. These solutions are incubated for h at 8 C, then diluted and their activity measured at 37 C, in the presence of increasing concentrations of compound n 2, by detection of l hydrolysis of p-nitrophenyl phosphate (PNP) at 1.3mmol / l, which results in

an increase in optical density to 405nm.

The results obtained again show the little influence of compound n 2 on the enzymatic activity of alkaline phosphatase as a function of the concentration (in mol / l) of compound n 2 in the medium.

of measurement.

Example 30: Renaturation of g-galatosidase.

In this example, we are investigating the possibility of renaturing 3galactosidase after it has been denatured by a protein solubilizer.

strongly denaturing consisting of urea.

A commercial suspension of 0-galactosidase is diluted 250 times in a solution containing 8mol / l of urea, 50mmol / l of dithiothreitol,

lmmol / l of MgCl2 and 50mmol / l of tris-

hydroxymethylaminomethane-HCl (pH 7.8). The solution is left for 30min at 20 ° C., then it is dialyzed for 18h against a 1mmol / l solution of MgCl2 and mmol / l of tris-hydroxymethylaminomethane-HCl (pH 7.8) in the presence of lmol / l of compound n 2 or in

the absence of this compound.

The enzymatic activity is then determined

as in example 24.

The results obtained (expressed as a percentage of the activity of the enzyme diluted in the same dialysis buffer without the addition of urea (control 1

and control 2), are given in Figure 8.

This figure also shows the activity obtained after dialysis when the dilution is carried out before dialysis with the same solution without urea, then dialysis with the solution of MgCl2 and of trishydroxymethyl amino methane-HCl without compound n 2

(control 1) and with 1 mol / 1 of compound 2 (control 2).

In this figure, it can be seen that 5.4% of the enzymatic activity is recovered in the absence of compound n 2 and that 11.8% of the enzymatic activity

are recovered in the presence of 1 mol / l of compound n 2.

Example 31: Separation by exchange chromatography

protein ions.

In this example, ion exchange chromatography is carried out on a MonoQHR5 / 5 column (Pharmacia Uppsala, Sweden). The starting buffer (buffer A) contains 20mmol / l of HEPES-NaOH (pH 7.4), 1mmol / l of ethylenediamine tetracetate. The limit buffer (buffer B) contains 20mmol / 1 of HEPES-NaOH (pH 7.4), 1mmol / l of ethylenediamine tetracetate and lmol / l

of NaCl.

The buffer flow rate is 1ml / min. The column is equilibrated with 10 ml of buffer A, then 0.1 Sml of a sample produced by digestion of fibronectin (0.5 mg / ml) with thermolysin is loaded as described by Sekiguchi et al in Journal of

Biological Chemistry, vol 260, no 8, 1985, p. 5105-

5114. Elution is carried out either by complex gradient from 0 to 100% of buffer B in A in 20 min, or by complex gradient from 0 to 100% of buffer B in buffer A in 20 min, the two buffers containing more

each 1 mol / l of compound no 2.

The fractions from the column are checked by analysis by gel electrophoresis on

polyacrylamide in the presence of sodium dodecyl sulphate.

FIG. 9 illustrates the results obtained during this analysis. The left part of this figure refers to the fractions obtained in the absence of compound n 2, while the right part refers to the

fractions obtained in the presence of compound n 2.

In this figure, we see that the fractions are identical, which confirms the absence of

disturbances in the presence of compound n 2.

TABLE I

Ex Compound Formula Compound nn H3

CH3 - '- CH2 - C2-

CH3 Irimcthylanmmonio methane carboxylale 2 n 2 2 / n 2 - (CH2) 3 - SO) 3 ** P-sf <.CC) N + - (CH2) 3 - su3- pyuidmium propane sulfonate 3 n 3 13 2-hydroxyethyl dimethyl ammonio propane 2 H 3 ul fonate nO4___ _HOCH2-CH2'- '(CH2) 3- S03- sulfoe 4 -n4 CH3 2-hydroxyethyl dimethyl ammonio butane HOC2'CH2-' _ * --- - (CH2) 4- S03 - sulfonate n S; H3 ethyl dimethyl ammonio propane sulfonate

_ n6 (C2i5- + - (CH2) 3-so3-

3 n6n * 6 (CH2) 3-S3 1-methyl piperidinium propane sulfonate

X_./- CH3

7 n 7 (CH2) 3-S03 '_ 0 J CH3 I-methyl morpholinium propane sulfonate

/ CH 3

8 n 8 3 cyclohexyl dinrethyl ammornio propane O + H- (CH2) 3 - S03- sulfonate l3 (C) 3'0 '

TABLE 2

EXTRACTION SOLUTION

* EX HEPES NaOH D'T PMSF SDS Triton Octyl Compound n 2 Compound n 3

X-100 G

9 10 mmnoU S m moII mm ol2moI / l 0.5 mmolA 10 g / - -

10 mmol / l 5 mmol / 2 mmolA 0.5 mmo / l 5 mlI -

i 10 mmol / 5 mnmolA 2 mmolAI 0, S mmo / I - 5 mLI - 200 g / l -

12 10 mmoll 5 mmnolA 2 mmolI 0.5 mmoUI - SUI. r 210 o

13 10 mmolU 5 mmol 2 mmoUl 0, S mmoI. 10o n -

14 0 mmoiUi 5 mmoUi 2 mmol 0.5 mmol / I -10 S / 200 8 / I -

s15 IO mmoi 5 mmol1 2 mmotI 0.5 mmotl 10 SA 210 o HEPES hydroxyethyl piperazine ethane sulfonic acid DTT = dithiothreitol PMSF = phenylmethyl sulfonyl fluoride SDS = sodium dodecylsulfate Triton X-100 = tert-octylphenyl- (oligo ethylene oxide) = octyl glucopyranoside

TABLE 3

DIALYSIS SOLUTION

EX | HEPES NaOH DIT EDTA KCI PMSF Compound Compound n 3 CHAPS n 2

16 10 mmol / I 5 mmoUl / 1 2 nunol / 1 mmol / l 23 mmol / 0, 5 mmoUl -

17 10 mmolA | mmol / I 2 mmoUlI mmol / I 2S mmoUIo / 0.5 mmol / I 200 g / l -

18 10 mnolI mmnol / I 2 mmnnol / I mmo / I 25 mmouI 0, 5 mmnunol / I- 210 S / 1 19 10 mmoUI / S maol / 2 mmol / II mmol / I 25 mmol / 0, S mmol / I - - 5 g / 10 mmoUlI mmo / I 2 mmol / I! mmolI 25 mmol / lO, S mmol / I 200 g / 1 5 g / 1 21 10 mmoUlI 5 nunoUI 2 mmol / II mnol / I 2S mmoIl 0.5 mnou / l 210 g / I 5 g / l HEPES hydroxyethyl piperazine acid ethane sulfonic DTT = dithiothreitol EDTA = ethylenediaminetetraacetic acid PMSF = phenylmethyl sulfonyl fluoride CHAPS = 3 (3cholamidopropyl) dimethyl ammonio propane sulfonate

Claims (20)

1. Agent for solubilizing biomolecules, characterized in that it consists of a non-detergent zwitterion corresponding to one of the following formulas: Ri RZ- + Y (I) or R X + B Y- (I R3 in which: - X represents N or P, -B represents a saturated hydrocarbon chain of 1 to 6 carbon atoms optionally comprising in its chain one or more oxygen and / or sulfur atoms, which may also contain one or more substituents consisting of OH groups, - R1, R2 and R3, which may be the same or different, represent hydrocarbon, aliphatic, cyclic or aromatic groups, having in addition 7 carbon atoms, including or not comprising one or more groups -O-, -Set / or -OH, Rl and R2 can also together form a saturated hydrocarbon chain of 4 to 8 carbon atoms, optionally comprising one or more OH substitutes and / or one or more oxygen and / or sulfur atoms, - R represents with X an unsaturated heterocycle with one or more rings containing at most 9 carbon atoms and one or more heteroatoms chosen from O, S, N and P, and optionally comprising one or more OH substituents, and - Y represents SO3-, OSO3- or COO-. 2. Agent for solubillization of biomolecules according to claim 1, characterized in that B is a hydrocarbon chain corresponding to one of the following formulas: - (CH2) 1, <(CH2) m-O- (CH2) n - (CH2) m-S- (CH2) n-, and - (CH2) m-CHOH- (CH2) n where 1, m and n are such that 1 <1 <6, 1 <m <5, 1 <n <5, and 2 <m + n <6 3. Agent for solubilizing biomolecules according to claim 2, characterized in that B represents - (CH2) 1 with 1 being equal to 3 or 4. 4. Biomolecule solubilizer according to any one of claims 1 to 3, characterized in that R1, R2 and R3 are chosen from methyl, ethyl, hydroxyethyl and cyclohexyl groups. 5. Biomolecule solubilizer according to any one of claims 1 to 3, characterized in that R1 and R2 together with X form a piperidino or morpholino group, and R3 represents a methyl group. 6. Agent for solubilizing biomolecules, characterized in that it corresponds to formula (II) with R and X + together forming the pyridinium group. 7. Biomolecule solubilizer according to claim 1, characterized in that it is chosen from trimethylammonomethane carboxylate, pyridinium propanesulfonate, 2-hydroxyethyl dimethylammonio propane sulfonate, 2-hydroxyethyl dimethylammonio butane sulfonate, ethyl dimethylammonio propane sulfonate, 1-methyl piperidinium propane sulfonate, 1-methyl morpholinium propane sulfonate and cyclohexyl dimethylammonio propane sulfonate. 8. Solubilizer according to one any one of claims 1 to 7, characterized in that that biomolecules are proteins. 9. Ethyl dimethylammonio propane sulfonate. 10. Methyl piperidinium propane sulfonate. 11. Cyclohexyl dimethyl ammonio propane sulfonate. 12. A method of solubilizing, or purifying, or separating or extracting biomolecules, characterized in that the biomolecules are brought into contact with a solution comprising at least one solubilizing agent according to any one of the following. claims 1 to 7. 13. The method of claim 12, characterized in that the solution comprises at least one another non-denaturing solubilizer. 14. The method of claim 13, characterized in that the other solubilizing agent is tert-octylphenyl- (oligo ethylene oxide) or octyl glucopyranoside. 15. Process for the purification or separation of biomolecules by electrophoresis, characterized in that one adds to the solution containing the biomolecules to be separated or to be purified, and / or to the separation medium, at least one biomolecule solubilizer according to any one of claims 1 to 7. 16. The method of claim 15, characterized in that the electrophoresis being gel electrophoresis, the agent (s) for solubilizing biomolecules is added to the solution of preparation and / or the rehydration solution of the gel. 17. The method of claim 15, characterized in that the electrophoresis being liquid stream electrophoresis, the (the) is added. solubilizing agent (s) in conductive solutions. 18. Process for the separation or purification of biomolecules by chromatography, characterized in that one adds to the solution of biomolecules to be separated, and / or to the conditioning and / or elution solutions, at least one biomolecule solubilizer. according to any one of claims 1 to 7. 19. Process for the renaturation of denatured biomolecules, characterized in that one adds at least one solubilizing agent of biomolecules according to any one of the claims 1 to 7 to the solutions used in the renaturation process. 20. Method according to any one of claims 12 to 19, characterized in that the biomolecules are proteins.