DE1935711B2 - Prepn of water-insoluble proteins bound - to mixed polymers - Google Patents

Abstract

Water-soluble proteins may be bound to mixed polymers to form water-insoluble products. The mixed polymers consist of (a) acrylamide, (b) ethenylmaleic acid or its anhydride, (c) maleic acid or -anhydride and (d) N,N'-methylenebis(acryl-amide) or ethylenediacrylate in a weight ratio of a:b:c:d = 3:0.1-1.5 : 0.05-4 : 0.075 - 0.9 where the sum total of (b) and (c) is a maximum of 4. Method may be used for enzymatic reactions and isolation of antigens and antibodies.

Description

The invention relates to a water-insoluble, carrier-bound protein.

A basic distinction must be made between insoluble proteins and bound insoluble proteins will.

It is known that insoluble proteins can arise from condensation, coupling and polymerization reactions with the protein itself. The proteins obtained in this way are products of uncontrollable reactions; there are none defined products. A large part of the protein is produced by the harsh reaction conditions the polymerization, the coupling with bis-diazo compounds and by the condensation Components used, such as formaldehyde, ethyl chloroformate and others, denatured and thereby biologically inactivated. It is lost for the main areas of application of insoluble proteins.

The only insoluble, biologically active proteins are the proteins attached to the carrier materials Significance because they represent defined materials that are universal for enzymes, inhibitors, antigens or antibodies, for protein substances in general,

The binding of the proteins to the carrier materials can be heteropolar or also homeopolar. The heteropolar bound proteins are of less interest because they depend on the ion concentration and pH can be removed again.

The following attempts to fix proteins to insoluble carriers using reactive groups are known:

1. Conversion of cellulose with p-nitrobenzyl chloride to the corresponding cellulose derivative, which is reduced to the amino compound and diazotized, coupled with proteins could.

2. The reactive group according to 1) was replaced by m-aminobenzyloxymethyl. Later attempts were made with

3. Polyamino-polystyrene - a fully synthetic carrier - · undertaken.

4. d.I-p-aminophenylalanine, d, l-leucine, and others. Ammo acids were polycondensed to a synthetic inert, whose diazotized amino Ability to couple groups with proteins

5. According to the methods of peptide chemistry, carboxy methyl cellulose with proteins and di-Cyciohexyl-carbodiimide implemented as a condensing agent.

6. The azide was produced from carboxymethyl cellulose via the hydrazide, and that with proteins able to react.

7. With bromoacetyl cellulose, proteins are well bound.

8. From isothiocyanate derivatives of dextran gel "Sephadex" and cellulose became water-insoluble enzyme compounds with proteins manufactured.

9. Under the conditions of acrylamide polymerization proteins can be fixed in easily swellable material. The enzyme is bound to the chains in a free-radical manner.

10. Next, proteins were bound to ethylene maleic anhydride (EMA) and

11. Styrene-maleic anhydride copolymers bound.

35 The previously known methods are unsatisfactory and cannot be generally used for proteins. Less than 50% protein is bound in methods 1, 2 and 4, and a large part of the protein loses its biological activity under these conditions. Method 3 turned out to be even worse.

Method 5 can only be carried out in a non-aqueous medium and therefore only partially for antibody problems and apply low molecular weight inhibitors. According to method 6, about 10% of the used Protein bound, of which only 10 to 43% retain enzymatic activity.

Method 7 gives a high yield of bound P.Otein (80 to 90%); for many proteins However, these reaction conditions mean a loss of biological activity.

With method 8, only a very small percentage of the protein used is actively bound to the gel.

According to method 9, only 18 to 20% of the protein used is bound, of which only a few percent Can develop activity towards substrates, substrates of higher molecular weight (hemoglobin) only become converted to 1 to 2%.

Currently, method 10 gives the most satisfactory results. It finds for antigens, inhibitors and enzymes Use. The binding reaction takes place gently in an aqueous medium; about 50 to 60% of the protein can be fixed under optimal conditions.

However, the raw material used is very large

6s inconsistent, it has low molecular weight (partly soluble) and higher molecular weight (insoluble) fractions that neither separate nor separate in any other way permit. Therefore, only up to 60% protein is added to the

I 935

insoluble carrier bound, the rest goes as soluble Material lost. But the bound protein is not yet satisfactory either.

In the binding of the protein to the carrier (EMA), the protein has a comonomer function; it can be bound not just in one place, but in several (ie networked). Depending on the protein concentration, more or less swellable protein-on-carrier substances are obtained. The protein is covalently bound in a network, so that diffusion plays an essential role in this material and it is only partially accessible to substrates. This is also achieved by adding various crosslinking agents (hexamethylenediamine, etc.). Disadvantage not canceled. Therefore, only about 50 ° _{o of} the bound protein can be achieved for low molecular weight substrates, so that the total activity is no more than 30 ° _o . Compared to high-molecular substrates, only 10 ° _o and less are found. " _J0

An additional disadvantage of this material is that it differs from protein to protein, even in Depending on the protein concentration, e.g. B. ilockig, difficult to nitrate and not without further can be packed in columns.

Stol-maleic anhydride polymers according to method 11 can be used to remove proteins, z. B. Purification of drinking water and others. These carriers are similar to polyaminopolystyrene pronounced lypophilic character. Proteins cannot be bound without losing their structure also loaded with proteins could not be lyophilized.

The functional groups of the known carrier substances that are not linked by protein carry _{positive or negative charges as NH 2} (method 1, 2, 3, 4) or as CO. Groups (method 6, 8, 10) in the corresponding pH ranges . In method 10, CO ₂ groups are also formed during the protein binding of the cyclic anhydride. This polyvalent character of the protein carriers causes adsorption of the matrix, similar to ion exchangers, increased inactivation or denaturation of the protein structure and shifting of the pH optima in the case of bound enzymes.

The object of the invention is to create a water-insoluble, Carrier-bound protein, which does not or only reduces the disadvantages outlined above having.

The present problems are solved by the invention. The invention is a water-insoluble, carrier-bound protein which is characterized in that it has been produced is off

55 A. a copolymer consisting of

a) acrylamide,

b) ethylene maleic acid or its anhydride and / or

c) maleic acid as well

d) Ν, Ν'-methylene-bis-acrylamide or ethylene diacrylate in a weight ratio a: b: c: d of 3: 0.5 to 1.5: 0.05 to 4: 0.075 to 0.9, the sum of b is) and c) at most 4 ratio of play, which was cyclized by heating in vacuo at 80 to 120 ⁰ C or at atmospheric pressure at 160 to 220 ° C, and

B. a biologically active protein by mixing either in aqueous solution and then Elution with saline solution or in aqueous saline solution.

The polymerized dicarboxylic acid is preferably located as anhydride. The component d), d. H. N.N'-methylene-bis-acrylamide or, and ethylene diacrylate, preferably makes up no more than 0.45 parts in the weight ratio given above

Acrylamide. N, N'-methyl-bis-acrylamide and / or Ethylene diacrylate and maleic acid or ethylene maleic acid or the corresponding anhydrides in the presence of radical formers and radical accelerators in the proportions given above polymerized by methods known per se. In itself it is possible. Maleic acid or maleic anhydride groups polymerize in broad concentration ranges. With regard to the task on which the invention is based, however, it turned out the composition described in more detail above as a condition for achieving a copolymer, which as a carrier substance has the desired favorable properties.

During production, the quantitative ratio of acrylamide to maleic acid or ethylene maleic acid can be determined or the corresponding anhydrides vary in relatively wide ranges without this has a significant effect on the amount of crosslinker required. So acrylamide can be mixed with maleic acid or their anhydride in a weight ratio of 3: 0.1 to 4, without this The ratio of acrylamide to crosslinker is significantly influenced. When using ethylene maleic acid or their anhydride as a comonomer, however, the limits are drawn more narrowly. Here the crosslinker content regulates pore size and strength, but also the binding of ethylene maleic acid in the gel.

For the fixation of proteins, particularly good results were obtained when using acrylamide and maleic acid or ethylene maleic acid or the corresponding anhydrides in a weight ratio 3: 0.5 to 1.0. When the concentration of the dicarboxylic acid is increased to 1.5, the copolymer becomes although more brittle and less swellable, it still shows good properties as a carrier. At a relationship from acrylamide to crosslinker, d. H. N, N'-methylene-bis-acrylamide or ethylene diacrylate, from 3: 0.075 to 0.450, gel-like polymers are used obtained particularly preferred properties. A ratio of 3: 0.075 represents the lower limit concentration represents, in which the maleic acid or ethylene maleic acid is still included satisfactorily.

Crosslinker concentrations have also proven to be useful for fixing proteins in an aqueous medium corresponding to a ratio of acrylamide to crosslinker of more than 3: 0.45, e.g. B. 3: 0.6 to 0.9, still considered well suited. When working with higher salt concentrations, i. H. more than 0.05M However, the relatively tightly crosslinked gels obtained in this way prove to be disadvantageous for the protein structure.

The copolymers can be prepared, for example, in aqueous solution, preferably in inert atmosphere. Examples of suitable radical formers or radical accelerators are Propionic acid nitrile and ammoniumproxydisulfnt. The polymerization can take place at room temperature, however

s 6

also takes place at higher temperatures up to 10O ¹ C or with solvolysis of the cyclic anhydride groups, can be carried out at a lower temperature. The protein structure remains after binding of the polymerization time int of the selected poly-protein, with activity yields up to the merization temperature, the selected accelerators 100% (and even above by increasing the catalyst or catalysts and the quantitative activity). The non-bound composition of the approach depends. Protein can usually be recovered between about 5 minutes and several minutes without loss of activity. When binding, the protein does not take over stunddi. a cross-linking function in the matrix, but rather the

After solidification, the polymerized batch obtained is called “enzyme gels” and is washed with water and tration different strength.
lyophilized. Through the targeted crosslinking, the moles can be For fixing the protein, it is necessary to adjust the molecular sieve properties of the copolymer so that the dicarboxylic acid is in the form of the anhydride 15 so that proteins are only present in the desired form. Areas must therefore be able to reach before the protein linkage. It is thus possible for all acid groups to be converted back into the cyclic interpolymer for certain proteins in this way to form hydride. This occurs for example crosslink, the protein binding surface only to de ^r top By heating the lyophilized copolymer can erMgen of the gel particle, but optionally in vacuum to a temperature of 80 to 120 ° C for 20 even throughout the gel. The polymerization parameters or under atmospheric pressure at ... one temperature can vary, depending on the effective molecular radius, from 160 to 220, preferably 180 to 200 ³ C. steric effects and charge distribution.

The copolymers are dependent on The copolymers have adsorption properties Crosslinker concentration water-clear to milky, so that in water or weak buffer turbidity substances with rubbery to brittle solutions (up to about 0.01 M) the protein 100% Consistency. Carrier is fixed. Up to 85% of the protein will be

The copolymers can be easily granulated, in this case covalently, the remainder being heteropolar. at for example by passing them through a metal sieve using the copolymers without prior use presses. Protein linkage is beneficial for the purpose of protein cyclization of the acid groups In this case, grain sizes of around 30 were found to be only adsorptive. It was found that 0.35 to 0.8 mm. These granules swell the protein with salt water when added under these conditions after. The strength of the swelling is due to the dissolution eluting from the carrier without loss of activity wetting content dependent. The closer the invention can.

Copolymers are crosslinked, the less swell- The protein binding capacity of the copolymers

they are bar. 35 sats depends on the number of available

The swelling is z. B. with alcohol or anhydride groups and the pore size. At a

easier by freeze drying. more closely networked product is the binding capacity

In some respects the particles are the same as those of a less closely crosslinked product

copolymers to be used according to the invention with the same number of anhydride

those of the known polyacrylamide molecular sieve. 40 groups because the protein molecule! with closer networking

In the case of treatment with buffer solutions, not all of the available bindable solutions occur

Shrinkage depending on the number of pre-groups can reach.

Existing carboxyl groups, similar to the ones in the mixed ion exchangers to be used according to the invention Too many charges on dextran or polyacrylamide polymer that are not from Base. 45 protein can be occupied by covalent bonding

If the carboxyl groups can be about by heating, a shift generally occurs 200 ° C can be converted into the cyclic anhydride, the pH optimum for the bound enzymes. Escapes often a slight yellow coloration. Is longer speaking was with the known carrier-bound Heated for time, the color deepens from yellow to enzymes observed that it is negatively charged brown, and at the same time the binding capacity increases 50 carrier materials to a shift of two clearly. A longer period of heating to the pH units occurs, for example at a temperature required for ethylene under normal pressure, maleic anhydride-bound trypsin of 7.8 therefore avoided as much as possible. to 10.0, and one for positively positioned carriers

The porosity and hardness of the copolymer shift into the acidic range. In the case of the gel in the state of a swollen mixed polymer, it is possible to determine the bondable line by the crosslinker content. The group egg should be used to the maximum, so that the carrier material has molecular sieve properties, ahn-bound proteins can be obtained, which are not lent as with the polyacrylamide gels, the shift in the pH value optimum
Any final limits due to the crosslinker content. A particularly interesting property that can be defined in accordance with the invention. In the swollen state 60 water bound to the carrier according to the invention is not easy to filter, or soluble proteins consist in the fact that under certain circumstances centrifugation and shows ideally higher Michaelis constants as column filling than in the case of properties. Unbound enzyme in water or buffer solutions. For example, swelling and de-swelling as a lyophilizate has been found to be gel-like in that the activity of the copolymer according to the invention can be retained without restriction. 65 bound trypsin versus M-benzoyl-1-arginine

The mixed polymer enables gentle p-nitranilid to be increased compared to unbound

Binding of the protein because it is hydrophilic and strong denem enzyme,

is swellable and the protein linkage is only possible by the carrier-bound enzymes according to the invention

have an abundance of highly desirable properties and can be used for numerous areas of application use. In this way, they make it possible to carry out substrate sales in a much more targeted manner than before, since that Enzyme can be removed from the incubation mixture at any desired point in time. is If the conversion is not yet quantitative, the same enzyme can be added again. With proteases that can protein to be cleaved can be isolated at any desired rate of hydrolysis. The cleavage reaction can track it directly. The enzymatically active protein no longer has to, as before, after the substrate turnover be precipitated - this often changed the substrate sales - but it can by simply filtering or decanting or the like. Remove.

Substrate sales can also be achieved with such carrier-bound enzymatically active proteins Run pillars. Carrier enzymes according to the invention packed in a cage-like vessel can be similarly carried out like a tea infuser add to an incubation mixture and remove from it at any time.

With proteolytically active, carrier-bound enzymes, inhibitors can be activated in a simple manner isolate. The stability of the proteases is significantly increased by the binding to the carrier material, since the proteases are no longer subject to an autolysis reaction.

With inertly bound protease inhibitors according to the invention proteases can be isolated easily, quickly and specifically. With their help it is too possible to purify protein solutions containing proteases from the proteases and thus to stabilize them. Simple isolation can be achieved by means of antigens or antibodies according to the invention which are fixed on carriers ίο from antigens or antibodies. With such carrier-bound enzyme antibodies can a particularly simple and effective enzyme isolation can be carried out.

The bound proteins according to the invention can generally be used and shown repeatedly partly no loss of activity even with prolonged use.

Due to the molecular sieve properties of the copolymers, the carrier-bound Also use proteins for substrate separation.

The copolymers are suitable for fixing biologically active proteins superior to the previously known carrier materials used for the same purposes to a large extent, as shown by the results summarized in the table below.

Carrier material uniformity
of the material Carrier: protein yield
born protein
in% Activity ⁴ )
low molecular weight
Substrates
in % Yield activity
of inserted
in% PAAD ¹ )
CMC HYDRAZID
(ENZYME) ² ) ...
EMA ³ )
according to the invention ... uniformly
uniformly
not uniform
uniformly 20: 1
10: 0.5 to 1
5: 1
any 1: 2 15 to 18
25 to 30
40 to 45
up to 80 90 ^s )
up to 40 ")
60 to 70 ')
up to lOO ⁸ ) 10 to 13
until 14
until 30
up to 80

') Polyacrylamide.

^e ) carboxymethyl cellulose hydrazide.
^s ) ethylene maleic anhydride.
·) Trypsin activity.

The superiority of the water-insoluble, carrier-bound proteins according to the invention is not only there in their superior yield and activity, but also in the far more advantageous permissible ratio from carrier to protein.

The water-insoluble, carrier-bound copolymers according to the invention are very capable of proteins different molecule size and properties, as the properties of this carrier material can easily be adjusted depending on the requirements of the protein to be bound. if 2. B. if the protein has a particularly large molecule, then by reducing the degree of crosslinking to a large extent, the penetration of the Protein into the matrix and binding in the same. For protein molecules that are so large that even by cross-linking to the lowest possible extent, their penetration into the matrix is made significantly more difficult it is also possible to fix the protein only on the surface of the particles undertake, in which case the copolymer is preferably so finely grained that a very large surface is obtained, the fixation then taking place solely on the surface of the protein leads to a completely sufficient degree of fixation.

⁵ ) all low molecular weight amino acid substrates.
^B ) Chymotrypsin Activity.
') Amino acid esters protected on nitrogen.
^! ) N-Benzoyl-1-arginine-p-nitroanilide.

The ratio of homeopolar to adsorptively or heteropolarly bound enzyme on the carrier can be determined influence by the way in which the bond between protein and carrier is established. For example the protein presented in a relatively high buffer concentration and slowly added the non-quenched gel, so the yield of homeopolar bound enzyme is very high and practically no adsorptive detect bound enzyme. On the other hand, if you work with a low buffer concentration and When carrier and enzyme are brought together quickly, the yield of bound protein is higher However, this protein, which has been transferred to the carrier, can be recovered with high buffer concentrations elute a considerable proportion, which can therefore not have been bound homeopolai. For example As stated above, working with a high buffer concentration and slow addition of carrier, this is how it works 80% of the enzyme presented to the carrier, and nothing more can then be eluted from it. There The enzyme is almost completely bound homeopoly, and its activity is only reduced the accessibility for the substrate is determined. In the second case, however, at low buffer concentrations and rapid addition, 100% of the submitted] protein is absorbed on the carrier. Let go of this

309585/42

9 10

but up to 35% again with high salt or buffer are with Ig carrier in 15 ml of ice-cold water,

Concentrations elute, so they were not homeopo-pH = 5.2, stirred in and kept at 40 ° C for 15 hours

lar bound. Refrigerator left to react. Then the

The following examples illustrate the invention. Product packed in a column.

Further. 5 The heteropolar bound protein was with

Preparation of the mixed polymer ^{65 μm] of} washing water eluted. With 0.2 M phosphate buffer,

pH = 7.8, no further protein could then be used

A. 3 g of acrylamide (AAD), 0.3 g of Ν, Ν'-methylene are eluted.

bis-acrylamide (nis-AAD) are bound with Ig ethylene heteropolar protein 31%

maleic anhydride (EMA) in 23 ml 0.05 M phos- io beioH7 0 8 5 10 9f) ° /

phate buffer pH 7.6 suspended and with strong residual activity, 'related' to 'non-

Stirring in a nitrogen atmosphere at room temperature federal trypsin 50 ° /

each with 1 ml of 5 ^o / _o sodium propionitrile and 5% '/ °

Ammonium peroxydisulfate added. After 10 to.

15 minutes the mass solidifies to a gel-like 15 B e 1 s ρ 1 e I 2

Block. This is 60 minutes at room temperature. The product from E. is used as a carrier.

left to stand and then through a metal sieve with The carrier is as described in A. with 100 mg

a clear mesh size of 0.5 mm pressed. Trypsin reacted.

The granules obtained are mixed with about 5 liters of distilled unbound protein ... 307

Water washed. The wet weight is now ao _{activity of the} bound protein ""

^ ^g ",, it xi- u 1 · * · λ ^before treatment with 0.2 M buffer 1007

The swollen copolymer obtained in this way w.rd _{after treatment with} fa _{M buffer} '»

freeze-dried to de-swell. In dried ° ^{/ a}

State, the weight (yield) is 5.4 g. .

For the cyclization to the acid anhydride, 25 B e 1 s ρ 1 e I 3 After 1.5 to 2 hours at 200 ° C in the drying cabinet The same carrier as in Example 1 is heated, turns. 100 mg of carrier and 200 mg of trypsin will be used

B. 3 g of acrylamide, 0.075 g of ethylene diacrylate and allowed to react as described in A.

1 g of maleic acid is mixed as described in A. Unbound protein 50 »/

polymerized. The polymerization time is 30% activity of the bound protein

¹⁵ J>. ^do . ^d , ^en ·,. . . ",. ... . . before treatment with 0.2 M buffer 100 ° /

Wener processing is also carried out as in A _after treatment with 0.2 M 48 V buffer

described. The yield of lyophilizate is °

2.3 g. After the cyclization, the weight of the “. ...

Product 2.05 g. 35 Example 4

C. 3 g of acrylamide, 0.3 g of N, N'-methylene-bis-acrylic- The product from C. is used as a carrier, amide and 1 g of maleic acid are as described in A. 100 mg of bovine serum albumin with 1 g of carrier ben copolymerized. Allow the polymerization time to react as described in A.

is 15 hours. Further processing takes place.,. ,,,, _,

also as described in A. The yield of 40 Unbound Protein 32%

Lyophilizate is 2.67 g. After cyclization, neither with 0.2 M phosphate buffer, pH = 7.8, is still

the weight of the product 2.45 g. with 0.2 M glycine buffer, pH = 1.8, protein from

D. As described in A. 3 g of acrylamide, carrier, are eluted. The yield of homeopolar 0.6 g of Ν, Ν'-methylene-bis-acrylamide and 1 g of maleic-bound protein is 68%, based on acid copolymerized. The polymerization time 45% protein, and 100%, based on bound is 5 hours. Further processing takes place in protein.

also as described in A. The yield of when using human serum albumin

Lyophilizate is 2.8 g. After the cyclization, the body of bovine serum albumin will be the same

2.6 g of final product obtained. Result obtained.

E. As described in A. 3 g of acrylamide, 50

0.3 g of Ν, Ν'-methylene-bis-acrylamide and 0.1 g of maleic example 5 acid copolymerized for 5 hours. Further processing is also carried out as described in A. The product from C. is used as a carrier. The wet weight of the swollen product is 1 g of carrier is added in portions within 60 ml-53.5 G. After lyophilization, a dry 55 groove is added to 200 mg of trypsin dissolved in 18 ml of phosphate weight of 4.8 g obtained. The cyclization leads to buffer (0.1 M, pH = 7.8) added and 18 hours at an end product in a yield of 3.1 g. Allowed to react at 4 ° C.

. . Unbound protein 50%

B e 1 s ρ 1 e 1 1 ^ activity of the bound protein,

Production of the carrier-bound protein ° based on the activity used ... 40%

A cyclized copolymer of 3 g. 0.2, 0.33 or 0.5 M phosphate buffer cannot be used

Acrylamide, 0.15 g Ν, Ν'-methylene-bis-acrylamide and protein and no activity eluted from the support

1 g maleic acid used as a carrier. 100 mg of trypsin will be used.

Claims (1)

Claim: Water-insoluble, carrier-bound protein, characterized in that it is produced has been out A. a copolymer consisting of a) acrylamide, b) ethylene maleic acid or its anhydride and / or c) maleic acid as well d) N, N'-methylene-bis-acrylamide or ethylene diacrylate in weight ratio a: b: c: d from 3: 0.5 to 1.5: 0.05 to 4: 0.075 to 0.9, the sum of b) and c) being at most 4 proportions that can be achieved by heating in a vacuum 80 to 120 ⁰ C or atmospheric pressure was cyclized to 160 to 220 ° C, and B. a biologically active protein by mixing either in aqueous solution and subsequent elution with saline or in aqueous saline. 25th