DE2017571C3 - Process for improving the stability of enzymes - Google Patents

Description

from the living cells) are present or they are reconstituted.

The invention is therefore a method for improving the stability of enzymes, thereby is characterized in that the enzyme is cooled in the aqueous medium to below 10.0 C and then in quick succession (a) the enzyme in a closed, microwave-permeable Container in a treatment zone by a sicli moving atmosphere of a cooling gas for one Inactivation exposes too short a period of time to microwave energy, with the moving Atmosphere in direct contact with the for! Microwaves permeable walls of the container, but no direct contact with the enzyme and has a temperature below about 15.6 C after entering the processing zone, (b) at the end of this period of time, the exposure of the enzyme to microwave energy ceases, and that one (c) cooling the enzyme.

The circulation of the cooling gas is preferably stopped after the exposure is canceled to the Microwave energy continued for a period of time to provide at least some cooling. The Enzyme material is thus preferably cooled to below 10.0 C, with at least the the latter part of this cooling process can be achieved by other means, for example by that a container of the enzyme material with a cold liquid wedge, e.g. B. cold water, in contact brings. It is also preferred to have the receptacle of enzyme material as opposed to it during exposure of the microwave energy rotating at such a rate that during exposure at least one complete rotation of 360, preferably a plurality of rotations of 360 "C, takes place, to ensure the influence of microwave energy on all main walls of the container.

The invention also provides a blood serum, which, compared to untreated blood serum, has an improved stability against destruction owns.

It has been found that the above treatment is suitable for increasing the stability of the treated Enzyme against destruction, d. H. against permanent loss of activity, to be increased considerably. It it is known that enzymes are permanently deactivated If you can get them more drastic thermal treatments including a treatment Subjected with microwave energy. The treatment carried out according to the invention now prevents by the effects of pre-cooling and rapid post-cooling, in combination with the effects of the cooling gas during exposure to microwave radiation and by the limited extent the latter, inactivating the enzyme, while at the same time increasing its stability.

The stabilization taking place according to the invention is achieved very quickly - in seconds - and is easy and economical to carry out. The conditions can be for any enzyme or a Enzyme mixture can be standardized in order to obtain reproducible results from time to time put. Thus, each lot or approach can be carried out according to the methods specific to such an enzyme to be examined.

As far as is currently known, all enzymes can be improved in terms of their stability, if the process according to the invention is used. Enzymes are proteins, including Metallo-proteins and conjugated proteins that are produced by living cells. After generally accepted classification from Webb in Biochemical Engineering, D. Van Nostrand Co., Inc., Princeton, N.J., 1964 (see Encyclopedia of Chemical Technology, Kirk - O t h m a ι, Second Ed .. Vol. 8), enzymes are divided as follows:

1. Hydrolyzing enzymes --- proteases and peptidases, such as pepsin. Rennin and the like: carbohydrases, such as amylases, esterases, e.g. E. Lipascn, Phosphatases and the like; Urease: desaminasc etc .:

2. Transmission enzymes - dehydrogenases. how Lactic acid dehydrogenasc: oxidases: transaminases, such as glutamic acid - transaminases: Kinases such as creatine phosphokinase;

3. addition and subtraction enzymes such as aconitase, enolase, carboxyase and aldolase;

4. Isomerases - such as alanine racemase;

5. synthetases - such as glutamine synthetase; and 6. nucleases such as deoxyribonuclease.

As stated earlier, the procedure is of the invention particularly suitable for the treatment of the enzymes in blood serum, the phosphotases.

Contains transaminases, dehydrogenases and phosphokinase. Any enzymes can be extracted from the blood serum or they are all isolated and, after reconstitution, handled separately if necessary. One can also treat the blood serum as such. Blood serum.

which is the clear liquid that after removal of the cell elements (red and white cells and platelets) and the coagulation mechanism: u; (Fibrinogen) remaining from the whole blood is one of the most special preferred materials that can be treated according to the invention.

The enzyme treated according to the invention is in an aqueous medium; i.e., it is not dry. It can be in its natural solvent, concentrated or not concentrated form, or it can be made by suspension or dissolution in a previously prepared aqueous medium after it has been isolated from its natural solvent or was concentrated in it. The amount of the aqueous medium associated with the enzyme is not critical as long as there is enough water to moisten the enzyme. The crowd can be very good depending on the end use of the product or on handling considerations be made. So it is B. is often most convenient to use with an aqueous suspension or solution work.

As is well known, microwave energy is the electromagnetic wave energy of the wavelengths specified in the microwave region of the electromagnetic spectrum.

For the purposes of the invention, a microwave energy is in an intermediate region with a Frequency from about 1000 to about 5000, in particular from about 2000 to about 3000 MHz, is preferred. the Microwave energy is supplied from a suitable radio frequency source, e.g. B. a magnetron generated. A feature of the method of the invention is the

Pre-cooling of the enzyme material. So that should Material at the time, which is called the first time Microwave energy is applied, a temperature well below room temperature, i.e. H. about below 10.0C. Although it can actually be frozen as it is opposite after exposure the microwave energy thaws, there is no need for this measure, so that one im For better handling a temperature above freezing point is preferred. One temperature in the range of about 1.67 to about 7.22 C is particularly satisfactory. The enzyme material can outside of the treatment chamber or zone or it can be .within the treatment chamber or zone are pre-cooled by \ before touching down against the microwave energy a previous flow of the cooling gas is provided.

A further feature of the method of the invention is that the material ¹ to be treated is stored in a closed container during the treatment. The walls of the container can be made of the conventional, substantially gas-impermeable Paekmalerialien and z. B. made of glass, l'oiymethylacrylat. Polystyrene and polyethylene are made up. They are essentially gas-tight.

The container containing the enzyme is Halls in a larger Bchandlungskammer or zone, in which the microwave energy is so directed is that they penetrate the microwave permeable walls of the container and into the enzyme enter.

Another feature of the method ler invention is the circulation of the cooling gas through the treatment chamber or zone and around the walls of the container containing the enzyme material. The cooling gas used can be any essentially inert gas (that with the environment in the presence of Microwave oven not responding), which is the case the working temperatures is gaseous. In particular, I will :! Air, nitrogen and carbon dioxide into consideration drawn. Although other gases such as argon, helium, neon, krypton, xenon and ethylene oxide or their Mixtures and the like are equivalent, they are on Reason for their cost .: less desirable.

The temperature of the cooling gas, which enters the treatment / .onc should be below about 15.6 C, and preferably about ¹ 12,8'C. While temperatures as low as -17.8 C can be used, there is no benefit in going below about 6.67 C with the temperature. Indeed, at such low temperatures, freezing problems can arise if an enzyme material is left in the treatment zone which contains the cold gas for a long time after the source of microwave energy has been turned off. Temperatures of the incoming gas between about -1.11 "C and about 10.0 C have been found to be particularly suitable. The cooling gas is heated during its migration through the treatment zone, in particular through contact with the walls of the container containing the The heated gas is removed from the treatment zone to make way for the cooling gas entering. If the gas is recirculated for reuse, then its temperature must be reduced to the desired temperature for entry into the treatment zone.

Since the main function of the cooling gas is to keep the walls of the container at a temperature which is well below that of the enzyme material to be treated brings about a forced introduction of the cooling gas into the treatment chamber and along the walls of the container below at least some positive pressure (of at least something

ίο above aunosphere pressure - k) a more effective overall cooling with it, without certain areas of the walls being insufficiently cooled. Pressures as low as 0.21 · 10 -6 kg / cm ² have already been used. Pressures up to about 3.52 kg / cm ² can be appropriate. Air is particularly satisfactory at low positive pressures, while essentially oxygen-free gases, particularly nitrogen, are preferred at higher pressures. The exact duration of exposure to the microwave energy may vary somewhat depending on the particular enzyme being treated, the volume of the enzyme material, the concentration of the enzyme in the material and the energy of the microwave transmitter. In general, the amount of time required is directly proportional to the volume of the enzyme-containing material and the concentration of the enzyme therein, and is inversely proportional to the power of the microwave transmitter. It has been found that the exposure time should in each case be at least about 1 second. It has also been found that excessive exposure results in complete inactivation of the enzyme. Since this is not the purpose, the total exposure time is designed to be so short that no such complete inactivation takes place. Since this time span differs for the reasons given above, it may be necessary to carry out preliminary tests in order to determine the extent to which the respective enzyme can be exposed to the microwave energy without it being completely inactivated. Each enzyme has its own analysis so that it can easily be determined whether a treated sample thereof has been completely inactivated or not. In any case, the period of time is shorter than that which causes the temperature of the enzyme material to rise to the temperature of the permanent inactivation of the enzyme. Generally, the amount of time will be no greater than that required to cause the enzyme material to rise in temperature by about 70.4 to 72.2 "C (125 to 130" F). In most cases, the temperature rise will be about 55.5 to 66.6 C (about 100 to about 120 ° F). In the method of the invention, the enzyme can be exposed to microwave energy all at once or in multiple stages.

A feature of the preferred method of the invention lies in the fact that all Hauptwändc de; Container containing the enzyme material, directly dei Microwave energy can be presented. This is best done by the fact that when in use an upright bottle as a container, the bottle on its longitudinal (vertical) axis at least once (36O " and is preferably rotated several times while the bottle is exposed to microwave energy traveling in a generally horizontal direction d. H. in a general to the main walls de: Container vertical direction, is directed. Dadurcl is achieved that all the main side walls of the container

be presented directly to the microwave radiation, whether it is circular, square or rectangular or have a polygonal cross-section.

After exposure to the microwave energy For the desired period of time, the micro-energy is turned off and the enzyme material is cooled down quickly. In this regard, it is preferable to cool with the cooling gas as well continue after the microwave energy has been turned off to cool the treated enzyme material or quenching, which is preferably done to a temperature of at least about 28.4 ° C. This can be done in that the container of the enzyme material in the treatment zone through which the cooling gas is passed, is left even after the microwave transmitter has been turned off is. In the case of one, continuously moving Series of containers of the enzyme material can do this by moving them across the field of direct exposure to the microwaves is expanded while the flow of the cooling gas is continued in contact with the containers. The enzyme material can also be used in other ways be cooled, in addition to or instead of the procedure described above, for example in that the walls of the container with a cold liquid, e.g. B. cold water, in Bringing contact, or by placing the container in a refrigerator. In any case it is preferable that the treated enzyme material is Temperature below 10.0 "C to cool.

The invention is illustrated in the examples.

was about 48.9 ° C. After switching off the magnetron (after operating for 62 seconds) the circulation of the cold nitrogen gas was continued for a short period of time until the Solution had cooled to about 26.7 ° C. The rotation of the compilation was canceled, opened the chamber and removed the bottles from the chamber and immediately placed them in an ice-water bath cooled to 4.44 ° C. The treated ίο samples were used together with untreated control samples stored at different temperatures and examined from time to time, with the following Results obtained were:

¹ S treated material

4.44 "C- after 13 days:
after 20 days:

21.1 ° C - after 13 days:
after 20 days:

583 units / ml, 375 units / ml; 497 units / ml, 375 units / ml;

37.8 ° C - after 13 days: 520 units / ml, after 20 days: 400 units / ml.

Control attempt

4.44 ° C - after 1 week: 315 units / ml,

after 13 days: 0; 21.1 ° C - after 1 week: 0; 37.8 ° C - after 48 hours: 0.

Example 2 example 1

Lactic acid chydrogenase ("LDH") was dissolved in a phosphate buffered non-serum aqueous base (pH about 7.2) to give 627 units / ml. The solution was filtered through a bacterial filter. Small sterilized glass bottles (7 ml nominal capacity) were filled with the solution, stoppered, and cooled to 4.44 ^r C. 48 of these bottles were then transferred to a pressure chamber equipped with a 2 kW magnetron. This was connected to a 220 V AC source and sent microwave energy at about 2450 MHz into the chamber. The bottles were divided into groups of three and each group placed on a small individual turntable. All the small individual turntables were held on a larger turntable, so that when the larger turntable was turned over at 24 rpm, the smaller, individual turntables turned at 60 rpm; that is, the small, individual turntables rotated 2.5 times for each revolution of the large turntable. This combination of turntables was made from polymethyl methacrylate. Cold nitrogen gas was flowed into, through and out of the chamber at a pressure of 0.18 kg / cm ² . The inlet temperature was about 1.67 ° C. The magnetron was then put into operation for 62 seconds. The magnetron and associated wave guides were arranged so that arc microwave energy was directed horizontally against the side walls of the bottles. The maximum temperature reached by the solution, scrum glutamic acid oxaloacetic acid transaminase ("SGOT") was dissolved in a phosphate buffered, aqueous non-serum base (pH about 7.2) to give 37 units / ml. A bacterial filter was then filtered. The solution was bottled, treated, stored and examined as in Example 1. The following results were obtained:

Treated material

4.44 "

2i, r

³ C - after 13 days

after 20 days
^D C - after 13 days

after 20 days
37.8 ° C - after 13 days: 16 units / ml,

after 20 days: 3 units / ml.

26.7 units / ml, 20 units / ml; 13 units / ml, 10 units / ml;

Control attempt

4.44 ⁰ C - after 1 week: 0.21.1 "C - after 4 days: 0,
37.8 ⁰ C - after 48 hours: 0.

Example 3

5 mg deoxyribonuclease from ox pancreas was reconstituted with 1 ml distilled water and then diluted to 800 ml with distilled water. The solution was fi triert. The initial aclivity after filtration was 786 units / mg. Small sterilized glass bottle (7 ml nominal content) were filled with the solution, stoppered and cooled to 4.44 ° C. Different Samples (24 bottles per lot) were then as ii Example 1, but for different periods of time

one JnnU

l'rohc Exposure time
(Seconds) Pointed height
the sample A.
B.
C. 60
68
74 about 44.4 ° C
about 46.1 ⁰ C
about 50.0 ⁰ C

After removing from the chamber at about 26.7 "C and cooling to 4.44" C in an ice-water bath the treated samples became 21 days together with the untreated controls stored at 25.0 ° C. The optical density of all samples was then determined by the analytical method von K u η it z, M., J. Gen. Physiol., 33, 349 (1950) using a Beckman DU spectrophotometer examined with wavelengths in the ultraviolet range. The following results were obtained receive:

sample

A.
B.
Γ

Control attempt

Analysis* (Units / mg)

475
520
450
200

* Average of 2 bottles from each treatment, three separate samples from each bottle.

Example 4

Lactic acid dehydrogenase isoenzyme of liver fraction extracted from ox heart was dissolved in sterile distilled water at 1 unit / ml, and the solution was filtered through a bacterial filter. Small, sterilized glass bottles (7 ml nominal capacity) were filled with this solution, stopped and cooled to 4.44 ° C. Forty-eight of these bottles were then exposed to microwave energy in a cold stream of nitrogen. After removal from the chamber at a temperature of about 26.7 ⁰ C and after cooling to 4.44 ° C, the treated samples were, together with the untreated control samples stored at room temperature. The control samples became completely inactive after standing overnight, while the treated material was as active as the original material after standing for 3 weeks. At the end of these 3 months there was only a loss of activity of less than 10% in the treated material.

Example 5

Lactic acid dehydrogenase isoenzyme of the heart fraction, that had been extracted from the rabbit muscle was added to sterile, distilled water 1 unit / ml, and the solution was filtered through a bacterial filter. After filling and stopping the material was treated and stored as in Example 4. The control samples were completely inactive after standing overnight.

ίο

acts. The following results were obtained:

while the treated material was as active as the original material after standing for 3 weeks. At the end of these 3 months, the loss of activity in the treated samples was less than 10 ° / _n

Example 6

An aqueous amylase solution (1000 units / ml)

> o was prepared and filtered through a bacterial filter. 10 ml portions of it were divided into 6 small, sterilized ones Glass bottles (10 ml nominal capacity) were filled, which were then stopped. The 6 bottles were on this cooled to 4.44 "C, and 3 of these were transferred to a pressure chamber equipped with a 2 kW magnetron was set up. The latter was connected to a 200 V AC source and was transmitting microwave energy at about 2450 MHz into the chamber. Each bottle was on a small, individual one

ao turntable, each of which is on a larger turntable was held, so that when rotating the large turntable, the small, individual turntable and the bottles on it rotated. The compilation was over

»3 made of polymethyl methacrylate. The big turntable and the small, individual turntables were rotated at 30 rpm. Then it got cold Nitrogen gas at a pressure of 0.18 kg / cm * in and through the chamber and directed out. The inlet temperature was about 1.67 ° C. Then the magnetron became 7 seconds turned on. The magnetron and the associated wave guides were arranged in such a way that that the microwave energy in the horizontal direction

was directed against the side walls of the bottles. After switching off the magnetron (after 7 seconds) the cold nitrogen gas was allowed to continue circulating for a short time until the amylase solution in the bottles had cooled to about 26.7 ° C.

The assembly was then switched off and the bottles removed from the chamber and immediately cooled to 4.44 ° C in an ice-water bath. The same was done with 3 untreated Control bottles were carried out, which had been held at 4.44 ° C for 48 hours. All samples were then examined for their amylase activity. The following results were obtained (Average values):

Material treated in this way 800 units / ml

Control material 200 units / ml

Example 7

Fresh human blood serum was filtered through a bacterial filter and filled into sterile glass bottles 30 ml were poured into the individual bottles

give. The bottles were stoppered and cooled to 4.44 ° C. Of these bottles, 3 were then in flowing nitrogen gas under the pressure of Example 6, but exposed to microwave energy for 15 seconds. After taking it out from the chamber at about 22 pounds to 23.9 ° C were the Bottles were immediately cooled to 4.44 ° C. in an ice-water bath. The bottles were then treated with untreated Control samples stored at 4.44 ° C and

11th

examined from time to time. At the end of these two weeks, the biochemical analysis values of the control samples had changed so that they were no longer within the normal ranges for the enzymes 12

lay. The treated material became tapered from time to time after continuous storage at 4.44 ° C Time examined over a period of 9 months. The following results were obtained:

table

component

30 values after the specified days ISO I

270

sodium

potassium

Glucose

Alkaline phosphatase ..

SGOT *

SGPT *

LDH * (total)

LDH fractionation

Isoenzymes liver fraction
(LDH ₁ )

Heart fraction (LDH ₅ ) ....
Remaining faction

Amylase

Creatine phosphokinase
(KPK)

Total protein

albumin

Globulin

upper limit average lower limit

upper limit average lower limit

upper limit average lower limit

upper limit average lower limit

upper limit average lower limit

upper limit average lower limit

upper limit average lower limit

upper limit average lower limit

obtre limit average lower limit

upper limit average lower limit

upper limit average lower limit

upper limit average lower limit

upper limit average lower limit

upper limit average lower limit

upper limit average lower limit

145 -

141.8

140

4.3 4.0 3.9

101 98.8 90.5

34.1 32.9 30.0

27.0 25.1 22.5

21.0 20.2 19.5

210 200 190

23.4 22.5 21.2

46.5 44.6 40.8

134

132.5

130.6

68 65 62.5

6.1 5.3 4.8

7.5 7.2 6.9

4.00 3.82 3.42

3.00 2.80 2.55 143

140.8 139

4.4 4.0 3.8

100.4 99.0 93.6

34.5 33.2 30.2

25.7 24.5 21.8

20.7 19.0 18.4

204
196
192.2

22.6 21.5 20.4

45.8 44.0 41.8

134

132.4 130.2

67
64
61.8

5.8 5.4 4.5

7.5 7.4 7.0

4.25 3.87 3.44

3.02 2.90 2.66

142

140.9

139.5

4.3 4.0 3.92

102 98.8 • 93.5

35.0 33.4 31.0

25.0 24.0 _21: 0

20.0 19.2 18.3

204 196 192

22.8 21.3 20.0

44.0 43.2 41.0

134 132 129.5

65

63.6

61.3

5.7 5.0 4.2

7.5 7.3 7.1

4.12 3.90 3.56

3.00 2.88 2.61

142.5

141

139.7

4.4 4.3 3.94

100 97.5 93.2

34.0 33.0 30.9

24.0 23.8 20.0

20.0 19.0 18.0

201 196 190

22, i 21.4 19.4

43.6 42.9 40.2

132

131.4

129.2

66.5 63.7 61.6

♦ *

7.5 7.3 7.0

4.19 3.87 3.51

2.94

. 2.91

2.60

14 ?.

141.5

139

4.4 4.3 3.8

100 97.5 91.4

34.1 33.0 30.5

23.5 22.8 20.0

19.0 18.7 17.8

198 190 188

22.5 21.2 19.0

43.1 42.6 40.3

132

130.2

128

65 62.6 61.4 **

7.4 7.3 7.0

4.11 3.92 3.70

2.98 2.90 2.70

* SGOT means seniral glutamic acid-oxaloacetic acid transaminase, SGPT means serural glutamic acid-pyruvic acid transaminase, LDH means mucic acid debydrogenase.

** No further tests were carried out

13th 14th

The treated material showed normal during the study period. ¹ , clctrophoretic behavior. The normal value; for these components are as follows:

Table 2

Components normal wcrtc

sodium

potassium

Glucose

Alkaline phosphate as ...

SGOT

SGPT

LDH (total)

LDH oil fraction

LDH-heart fraction

LDH remaining fractions

Amylase

Creatine Phosphokinase ...

Total protein

albumin

Globulin

135 to 145 meq. / L 3.5 to 5.0 meq. / L
65 to 110 mg ° / ₀

5 to 35 units (international) 5 to 40 units (international) 5 to 35 units (international) 150 to 500 units (international) 10 to 20 "/ o of the total 20 to 40% of the total 35 to 55% of the total 50 to 160 units (dialase) up to 35 units (international) 6.8 to 8.0 mg ° / ₀
3.25 to 5.70 gm / ₀ 1.5 to 3.0 gm "/ ₀

Claims (12)

Patent claims: 1. A method for improving the stability of enzymes, characterized in that that one cools the enzyme in the aqueous medium to below 10.0 C and then more rapidly Succession a) the enzyme in a closed, microwave-permeable container in a treatment zone by a moving atmosphere of a cooling gas, a period of microwave energy too short for inactivation exposing the moving atmosphere in direct contact with the microwave permeable walls of the container, but has no direct contact with the enzyme and after your Entry into the treatment zone has a temperature below about ί. \ 6 C, b) at ^E: .nde this period of time the exposure of the enzyme of the microwave energy breaks off and that c) the enzyme cools down. 2. The method according to claim 1, characterized in that the microwave energy has a frequency from about 1000 to about 5000 MHz. 3. The method according to claim 2, characterized in that that the microwave energy has a frequency of about 2000 to about 3000 MHz. 4. The method according to claim 3, characterized in that that the microwave energy has a frequency of about 2400 to about 2500 MHz. 5. The method according to any one of the preceding claims, characterized in that the treated enzyme in the container continues to act after exposure to microwave energy of the cooling gas. 6. The method according to any one of the preceding claims, characterized in that the enzyme in the aqueous medium is at least one of those in the blood serum. 7. The method according to any one of claims 1 to 5, characterized in that the enzyme is im aqueous medium is blood serum. 8. The method according to claim 7, characterized in that the blood serum before exposure the microwave energy cools to a temperature of about 1.67 to about 7.22C. 9. The method according to claim 7 or 8, characterized in that the temperature of the blood serum does not exceed 51.7 "C during exposure to microwave energy. Process according to one of Claims 7 to 9, characterized in that the blood serum is cooled ^{to below approximately 10.0 ° C. after exposure to the microwave energy.} 11. The method according to any one of the preceding claims, characterized in that substantially all of which are microwave transparent Main walls of the container are directly exposed to the microwave energy. 12. Blood serum, characterized in that it according to the method according to the pinem of claims 7 to 11 has been obtained. The invention relates to a method for improving the stability of enzymes. Enzymes are generally unstable in a liquid aqueous medium, regardless of whether : ■ they in their natural solvent (after removal from the living cell, which is the source for this forms) or after isolation from their natural solvent and after V. 'low suspension or Re-dissolution (reconstitution) in a previously ίο produced aqueous medium are present. Commercially available Enzymes, d. H. Enzymes produced for industrial purposes including medical purposes and put on the market normally irocknet, although there are also cases in which certain enzyme preparations are classified as co Syrup can be put on the market. During the time it takes to dry completely or concentration is required, some destruction of the enzymes may occur. For the end The dehydrated or concentrated enzymes are often used by suspension or Dissolution reconstituted in an aqueous medium. This reconstitution brings the possibility of errors with itself, especially if the reconstituted material is by weight or Volume unit must have a certain activity value. In addition, the enzyme is reconstituted subject to destruction, the speed and extent of which depends on various factors such as the Temperature, concentration, time and nature of the reconstructing medium, e.g. B. des pH value, depends. For example, automatic, electronic diagnostic methods for examining body fluids such as blood, blood plasma, blood serum and urine require a control blood serum that should be as close as possible to the fresh natural and normal blood serum. However, blood serum now contains many enzymes. Ice has therefore been necessary in the past to lyophilize the blood serum marketed for this purpose. The lyophilized material then had to be reconstituted with water before it could be used in the analytical methods mentioned. This procedure was associated with restrictions un ⁴ brought disadvantages. Lyophilization can already disturb the delicate balance of the components of the serum. The reconstitution of the lyophilized materials thus often led to serious errors. It has hitherto been necessary to produce a large number of different lyophilized blood serum products, from which one or more were then selected in accordance with the particular analysis to be carried out. In addition, the reconstituted material had a limited shelf life so that a fresh pack of lyophilized material would normally have to be reconstituted and used daily. The foregoing illustrates the problems caused by the instability of the enzyme preparations are conditional. These problems have been described in detail because the blood serum is a very sensitive one and is unstable enzyme material to which the method of the invention is particularly applicable is. The invention is also applicable to enzymes in general, regardless of whether they are in their natural solvent (after removal