CH626402A5 - Process for immobilising proteins on a solid support - Google Patents

Description

The present invention relates to a process for the immobilization of proteins on a solid support, characterized in that the support is reacted with a quinone to activate it, and that the subsequent reaction between the activated support and proteins takes place at temperatures between 0 ° C and the starting temperature of protein denaturation.

The activation reaction of the support can take place with the quinone as it is and with the quinone bound to a substance with a high molecular weight, in which case proteins linked to high polymers soluble and insoluble in water will be obtained.

The protein substances thus modified can be of considerable use in various fields of application depending on their functions. In particular, if the proiteic substances are enzymes, i.e. biological catalysts, once attached to water insoluble supports, they can be used as heterogeneous catalysts in the preparatory, analytical, biomedical fields.

As heterogeneous catalysts they become easily separable from the mixture in which they are put to react, and since it has been seen that after this chemical modification they surprisingly retain remarkable stability, and can be used repeatedly.

Even those enzymes whose isolation and purification procedure is very laborious and expensive, once modified, can be repeatedly used with considerable economic advantage.

Furthermore, the enzymes, according to the process as defined in claim 1, can be attached to water-soluble polymers. In this way their stability to denatured agents increases considerably and therefore they can be used in more drastic experimental conditions than those normally used.

The molecular weight of these enzymes linked to soluble polymers also increases with respect to that of the corresponding native enzymes, and thus allows their use in systems separated by ultrafiltrating membranes which allow only the reaction products to pass.

If the protein substances are antigens, once attached to water insoluble supports, they can be used in this form to selectively separate the corresponding antibody present in a complex mixture.

If protein substances are enzyme inhibitors, once attached to insoluble matrices, they can be useful for the selective separation of the enzymes that they inhibit.

In the event that the protein must be bound to a substance with a high molecular weight, it is then the latter that is previously treated with quinone.

The reaction between the support and the quinone is carried out at temperatures ranging from 0 ° C to 150 ° C, preferably around 20 ° C.

The incubation time varies according to the type of support and is generally comprised between 0.5 and 100 hours, while the reaction pH can vary between 1 and 11, preferably around 7.5.

The support to be activated can contain various types of reactive groups such as, for example, hydroxyl, amine, sulfhydryl, imidazole, pyrrolic, phenolic, guanidine, etc. The reaction can take place both in an aqueous and anhydrous medium depending on the quinone used and the type of polymer. Once the support is activated, the reaction is carried out with the protein.

It has been found, among other things, that these quinones can also lead to the cross-linking of protein molecules with each other, or to the formation of intramolecular cross bonds with consequent stabilization of the protein conformation.

The degree of crosslinking can be adjusted so as to obtain protein derivatives with a molecular weight higher than that of the native protein, still soluble in water, as well as protein derivatives crosslinked to such an extent as to become insoluble in water.

The characteristics and operating methods of the invention will be understandable by examining the following examples of execution which have a purely explanatory value.

Example 1

Immobilization of trypsin on sepharose 4B-AH with 1,4 benzoquinone

1 gram of Sepharose 4B-AH (Pharmacia Fine Chemicals AR-S-75104 Uppsala I-Sweden) is suspended in 20 ml of a 0.5 M aqueous solution of sodium chloride and left overnight. Then it is placed in a column (0.9X15 cm) and washed with 100 ml of this solution and finally with 50 ml of 0.05 M sodium phosphate buffer at pH - 7.4.

All the solid (4 ml) is suspended in 25 ml of this buffer and added with 2 ml of a freshly prepared 0.1 M aqueous solution of 1.4 benzoquinone. The mixture is left under stirring for 24 hours at room temperature in a container protected from light; the whole is then introduced into the column (0.9 X15 cm) and the solid is washed with 100 ml of the reaction buffer. This results in an activated solid support, intensely colored in dark brown. This is suspended in 25 ml of the same cold buffer and added with 5 ml of a solution of crystalline trypsin (British Drag Houses Ltd. Laboratory Chemicals Division - Poole, England) in cold water (10 mg / ml).

After 24 hours of contact at 2 ° C the whole is introduced again in the column (0.9 X15 cm) and the solid is washed alternating aliquots from 15 ml of 0.05 M sodium phosphate buffer at pH 7.4 and pH 4 , 4 for a total volume of 150 ml. A trypsin supported on 4B-AH is obtained which produces an esteric activity of 35 units per ml of support (the esteric activity was determined with N-benzoyl-argi-nin-ethyl ester (BAEE) as substrate, at 25 ° C epH = 8.0 according to the method of L. Goldstein, Methods in Enzymology -Vo. 19, 935-962 (1970). An enzymatic unit is that quantity of enzyme which hydrolyzes 1 limole of substrate per minute under the conditions specified above) .

This immobilized trypsin sample stored at 4 ° C, in 1 M aqueous solution in sodium chloride and 0.5 M in monosodium phosphate (pH — 4.4), occasionally retrying the supernatant solution, after three months it still carries out all the activity initial esterasic.

A 4B-AH sepharose sample not treated with chi-none, but in contact with the same amount of trypsin

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for the same time and subsequently washed it does not exhibit any enzymatic activity, demonstrating that there is no physical adsorption of enzyme on the support.

Example 2

Immobilization of trypsin on sepharose 4B with 1,4 benzoquinone

A 4 ml sample of Sepharose 4B (Pharmacia Fine Chemicals AB) is washed as described in the previous example, suspended in 25 ml of 0.05 M sodium phosphate buffer at pH = 7.4 and added with 2 ml of an aqueous solution. 0.1 M of 1.4 freshly prepared benzoquinone. It is left in a container protected from light, at room temperature, under stirring for 20 hours. Then the dark brown colored solid obtained is washed, reacted with trypsin and then washed exactly as described in the previous example. At the end a 4B sepharose containing trypsin is obtained which carries out an esteric activity of 26 units per ml of support.

This compound after three months of storage under the same conditions of example 1 still carries out 95% of the initial esteric activity.

Also in this case, a support sample not treated with quinone, but treated with the same amount of enzyme and subsequently washed, does not exhibit any enzymatic activity.

Example 3

Immobilization of trypsin on sepharose 4B with 1,4 benzoquinone

A 4 ml sample of sepharose 4B, washed as described in Example 1, is suspended in 25 ml of 0.05 M sodium phosphate buffer at pH = 10 and added with 10 ml of a fresh aqueous solution 0.1 M of 1 , 4 benzoquinone.

The mixture is left under stirring overnight at room temperature in a container protected from light. The activated solid is then transferred to the column (0.9 X15 cm) and washed with 200 ml of 0.05 M sodium phosphate buffer at pH 7.4; it is then reacted with the enzyme and washed following exactly the methods described in example 1. At the end a solid product is obtained which has an esteric activity of 65 units per ml.

Example 4

immobilization of trypsin on p-amino benzylcellulose with 1,4 benzoquinone

2 grams of para-amino benzylcellulose (0.11 meg / g) (British Drug-Houses Ltd. - Laboratory Chemicals Division) are suspended in water, introduced into a column (0.9 X15 cm) and subjected to three washing cycles, each of which consists of 50 ml of 0.1 N HCl in water, 20 ml H20, 50 ml of 0.1 N NaOH in water and 20 ml H20. Finally they are washed with 200 ml of 0.05 M sodium phosphate buffer at pH 7.4 and divided into two equal parts.

A part is suspended in 25 ml of this buffer, added with 4 ml of an aqueous solution, 0.1 M of 1,4 - benzoquinone freshly prepared, and left under stirring at room temperature for two days. Then the solid, colored in brown which is thus obtained, is washed in a column (0.9 X15 cm) with 150 ml of the same buffer. It is then suspended in 25 ml of this cold buffer and added with 5 ml of a cold aqueous solution of trypsin (10 mg / ml). The whole is kept under stirring at 4 ° C for two days. Finally the solid is re-washed in the column (0.9 X15 cm) with the reaction buffer until the washing waters no longer contain protein that can be measured spectrophotometrically at 280 nm. The product thus prepared has an esteric triptych activity of 55 units per gram.

The other part of p-aminobenzylcellulose washed, not treated with quinone, but treated with the enzyme and subsequently washed, using the same conditions described above, does not exhibit enzymatic activity.

Example 5

Penicillin-acylase immobilization on sepharose 4B with 1,4 benzoquinone

A sample of 4 ml of sepharose 4B, washed as described in Example 1, then with 100 ml of 0.05 M sodium phosphate buffer at pH 8.0 is suspended in 16 ml of this buffer and added with 5 ml of aqueous solution 0.1 M of 1.4 freshly prepared benzoquinone. The mixture is left under stirring at room temperature for 13 hours in a container protected from light.

The dark solid thus obtained is washed in a column (0.9 X15 cm) with 100 ml of buffer described above, finally suspended in 6 ml of buffer solution and added with 25 ml of a penicillin-acylase solution from Escherichia Coli ATCC 9637 in the same buffer (the enzyme was prepared according to Marconi et al. - Journal of Antibiotics Vol. 26 - page 228, 1973). The mixture is left under stirring at room temperature for 4 hours, then the whole is transferred to a column (0.9X15 cm) and the solid is washed alternating 15 ml aliquots of 0.05 M phosphate buffer and 1 M at pH 8.0. The total volume used for washing is 150 ml, of which only the first 50 ml contain protein that can be measured spectrophotometrically at 280 nm. A dark solid containing enzyme is thus obtained which has an activity of 125 units per ml.

125 ml of enzyme solution reacted with the solid contained 1400 units of enzyme with a specific activity of 21.1 units per mg, while the mother liquors of the reaction mixture and the usual combined washing waters contain 905 enzymatic units.

In practice all the enzyme put to react is recovered; moreover, the immobilized enzyme shows the same activity as when it is in solution.

A sample of sepharose 4B, not treated with quinone, but treated with the enzyme and washed following the same procedures described above does not show any enzymatic activity, demonstrating that under the conditions adopted there is no adsorption of enzyme on the support.

(The enzymatic activity was determined at 37 ° C using Penicillin G as a substrate at pH 8.0 according to the Marconi et al. Method, Journal of Antibiotics - Vol. 26, p. 228, 1973). An enzymatic unit is the amount of enzyme which hydrolyzes 1 penicillin G limole per minute under test conditions. Repeated uses of the immobilized enzyme performed over 1 month have shown irrelevant losses of activity.

Example 6

Penicillin-Acylase immobilization on sepharose 4B with 1,4 benzoquinone

A 4 ml sample of sepharose 4B is washed and activated with 1,4 benzoquinone following exactly the methods of the previous example. It is then reacted with 1580 penicillin-acylase units from E. Coli with specific activity 2.7 units per mg.

The reaction time is 15 hours, while the other modes and washes are the same as in the previous example.

1300 enzymatic units are recovered in the reaction and washing waters, while the solid exhibits an enzymatic activity of 67.5 units per ml.

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Note that in this case the method gives equally satisfactory results even if a poorly purified enzyme is used. Also in this case repeated uses of the immobilized enzyme carried out within 1 month showed irrelevant losses of activity.

Example 7

Immobilization of Penicillin-acylase on sepharose 4B-AH with 1,2 naphthoquinone

1 gram of 4B-AH sepharose, treated and washed with 0.5% NaCl aqueous solution described in Example 1, then washed with 100 ml of 0.05 M sodium phosphate buffer at pH 8.0, finally suspended in 25 ml of this buffer and added with 25 ml of dioxane containing 0.5 millimoles of 1,2-naphtho-quinone. The mixture is left under stirring at room temperature, in a container protected from light for 18 hours, then filtered, the solid is washed with 50 ml of a solution obtained by mixing a volume of 0.05 M phosphate buffer at pH 8.0 with a volume dioxane. Finally the solid, introduced into the column (0.9 X15 cm) is washed with 100 ml of buffer. The black product thus obtained is reacted with penicilli-na-acylase (from Escherichia Coli ATCC 9637). The reaction mixture contains 4 ml of 4B-AH sepharose activated at 2030 enzymatic units with a specific activity of 2.7 units per mg in 30 ml of 0.05 M phosphate buffer at pH 8.0. The whole is left under stirring for 1 hour at room temperature and 48 hours at 4 ° C. Then the solid is washed in the same way as in Example 5 and at the end an immobilized enzyme is obtained which performs an activity of 55 units per ml of support, while the enzymatic units recovered in the mother liquors and in the washing ones amount to 1815 units. Total. Also in this case there is no loss of enzyme and also the immobilized one performs the same activity as when it is in solution.

Example 8

Immobilization of peroxidase on sepharose 4B with 1,4-benzoquinone

A 4 ml sample of sepharose 4B is activated with 0.5 millimoles of 1,4-benzoquinone at pH 8.0 using the same methods as in example 5, finally treated with peroxidase (product grade I of the company Boehringer Mannheim GmbH, 6800 Mannheim 31 - Federal Germany). The reaction mixture contains 4 ml of activated 4B sepharose, 100 mg of enzyme and 0.05 M phosphate buffer at pH 6.0; the total volume is 30 ml. The mixture is left under stirring at 4 ° C for 24 hours, then washed alternating portions of 15 ml of 0.05 M and 0.5 M phosphate buffer at pH 6.0 for a total volume of 150 ml and finally the solid activity. It is 488 units per mi of support.

The peroxidase activity was determined with ABTS (2,2'-azino-di (3 ethylbenzothiazoline) -6 'sulfonic acid) (Boehringer Mannheim GmbH) and hydrogen peroxide at 25 ° C and pH 7.0 according to the method recommended by the Enzyme supplier company.

An enzymatic unit is the amount of enzyme that transforms 1 substrate limole per minute.

Example 9

Immobilization of anti Ts antibody with 1,4-benzoquinone on sepharose 4B

A 4 ml sample of sepharose 4B is activated with 0.5 millimoles of 1,4-benzoquinone as described in example 5, then reacted with anti-Ta (3,3 ', 5 tri-iodiothyronine) antiserum from rabbit . The reaction mixture contains 4 ml of activated 4B sepharose, 20 ml of antiserum, 0.2 M potassium phosphate buffer, 0.1 M KCl at pH 7.4. The total volume is 30 ml. The whole is kept under gentle stirring overnight at 4 ° C, finally the solid is filtered and washed with 200 ml of 0.02 M buffer in potassium phosphate and 0.05 M in trishydroxymethylaminomethane hydrochloride (Tris-HCl) at pH 7.4 and stored for 24 hours at 4 ° C in this buffer.

In parallel, the same operations are performed on a control blank, to which only the treatment with the antiserum is omitted. On the two solid substrates prepared in this way, the taking tests of the hormone 1 ^ are carried out:

0.6 ml of support (equal to about 20 mg of dry product) are balanced with 35 nanograms of Ta labeled with 123I in 1.5 ml total, containing 0.2 M phosphate buffer and 0.1 M KCl at pH 7, 4, for 1 hour at 37 ° C and overnight at 4 ° C. The liquid is separated from the solid by centrifugation and the radioactivity taken from the solid is measured.

The solid treated with the antiserum takes 58% of the total radioactivity set to balance, while the white takes only 11%.

Example 10

Immobilization of the trypsin inhibitor from soybeans on sepharose 4B with 1,4-benzoquinone

A sample of 4 ml of sepharose 4B are washed and activated with 0.5 millimoles of 1,4-benzoquinone as described in example 5; then reacted for 10 hours with 25 ml of 0.02 M sodium phosphate buffer at pH 8.0 containing 60 mg of soybean trypsin inhibitor (Boehringer Mannheim GmbH). The substrate was washed with 0.1 M KCl and 0.02 M sodium phosphate buffer at pH 8.0 until the washing water no longer contains protein. The pooled and analyzed mother liquors and washing waters contain 46 mg of residual protein. The support is then washed with 200 ml of pH 8.0 buffer containing 0.05 M Tris-HCl, 0.1 KCl and 0.02 M Ca Cl2 and left for 24 hours at 4 ° C in this buffer.

The 4 ml of compound thus prepared have the capacity (determined by the method of LJ Loeffler and JV Pierce - Biochimica et Biophysica Acta, Vol. 317, page 20, 1973) to bind 13.2 mg of crystalline trypsin with an efficiency of 63 % compared to the immobilizer inhibitor.

Example 11

Trypsin stabilization with 1,4-benzoquinone

50 mg of crystalline trypsin (British Drag Houses) are dissolved in 50 ml of 0.05 M sodium phosphate buffer pH 7.3 and added with 0.2 ml of a 0.1 M aqueous solution of 1.4-benzoquinone. The mixture is left under stirring at room temperature for 2 hours and then for 20 hours at 4 ° C. Finally the reaction mixture is cold dialyzed against water brought to pH 3.0 with dilute hydrochloric acid. All these operations are carried out in parallel also on a control blank, to which the addition of the quinone is omitted. Both the reacted solution and the white solution after dialysis are stored at 4 ° C and stability stability is tested on them. After 5 and 11 days, the enzymatic activity of trinine treated with quinone and untreated trypsin is determined. The treated trypsin shows 100% and 97% of the initial activity while the untreated trypsin 84% and 74% respectively.

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Claims (4)

626402 1. Process for the immobilization of proteins on a solid support, characterized in that the support is reacted with a quinone to activate it, and that the subsequent reaction between the activated support and proteins takes place at temperatures between 0 ° C and the temperature the start of protein denaturation. 2. Process according to claim 1, characterized in that the second reaction takes place at a temperature preferably chosen between 0 ° C and 25 ° C. 2 CLAIM 3. Process according to claim 1, characterized in that the second reaction is carried out at a pH variable from 3 to 11. 4. Process according to claim 1, characterized in that the support contains reactive groups selected from the hydroxyl, amine, sulfhydryl, imidazole, pyrrole, phenolic, sulphonic and guanidine groups.