CA1127055A - Stabilized liquid enzyme and coenzyme compositions and method of preparing same - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE Labile coenzymes, such as nicotinamide-adenine dinucleotide (NAD), are stabilized by treatment with an organic solvent, such as propylene glycol, in an aqueous media and a suitable polymer which does not inhibit enzymatic activity. An azide compound may be added to the solution, which not only serves as a bacterio-stat, but also functions as a stabilizer. In addition, a second coenzyme, such as adenosine triphosphate (ATP), may also be stabilized in the same solution. Moreover, one or more enzymes, such as hexokinase and glucose-6-phosphase dehydrogenase, may be stabilized against denaturation in the same solution. The composition exhibits excellent shelf life, and the container therefor may be repeatedly opened for use without any substantial degradation of the labile components. Moreover, all of the components may be packaged in a single solution. -1-

Description

'7~}55 This application is a divisional of co-pending application Serial Number 285,845, filed August 31, 1977.
1B ZiCKGRO~lN D OF THE INVE_T I ON

2 I. Field of the Invention 3This invention relates in general to certain new and 4 useful improvements in the stabilization of enz~nes and coenzymes and the method of stabilizing, and, more particularly, to sta-6 bilized labile enzymes and coenzymes in a single aqueous organic 7 solvent media.

9 II. Description of the Prior Art The present commercial state of the art used for stabilizing 11 the reactive ability of enzymes or coenzymes is by locking them 12 into a solid matrix, either by freeze drying, dry blending such 1~ ` as used for tableting dried powders, primarily in the pharmaceu-1~ j tical diagnostic and related industries and immobilization by 15 I locking the chemical structure of the enzyme into a solid matrix.
16 ¦ Contrary to the sophistication these terms imply, these approaches 17 ¦ are neither practical nor desirable and are also expensive.
18 The manufacturer is forced to remove the water and supply a 19 I partial product, thus relinquishing part of the quality control 20 I cycle in the dilution and use of the final product. Laboratories 21 I are forced to pay the high cost of packaging, reagent waste, 22 I freeze drying-and dry blending, and usefulness of the produce 23 I is ~urther limited by packaging modes and sizes.
24 ¦ Furthermore, good product uniformity is difficult to 25 ¦ achieve. This condition is exemplified by the fact that most 26 commercial freeze dried control sera (reference serum) list the 27 I acceptable bottle-to-bottle variation of enzyme constituents at 2~ I + 10% of the mean.
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2 It is, thereEore, the primary object of the present inven-

3 tion to provide a liquid composition with coenzymes and/or enzymes which are stabilized in a single container.
-5 It is a further object of the present invention to provide 6 a labile enzyme and coenzyme composition of the type stated in an aqueous organic solvent media and where the stabilization of 8 the enzyme and coenzyme does not affect the enzymatic reactivity 9 after a substantial period of time.
~ ~ 10 It is another salient object of the present invention to ,;11 provide a method of stabilizing labile enzymes and/or coenzymes 12 in the presence of other labile coenzymes or otherwise other 13 labile enzymes and which composition has a long shelf life.
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SUMM~RY OF THE INVENTION
_ Labile enzymes and coenzymes are trea-ted accordiny to the invention, resulting in long-term stability without aEfezting enzymatic or coenzymatic reactivity or phometric absorptivity.
Providing enzyme and coenzyme reagents in a stable liquid form enhances -the colorime-tric applicability of present day NA~/NADH
coupled methodologies, as well as other methodologies, primarily because the separation oE ingredients is easily accomplished. Stable liquid reagents are especially advantageous where NADH and other coenzyme consumption is the basis of measurement and the color reagent must be separated from NADH and the reaction main. In the ultraviolet mode, the liquid system offers better reagent homogeneity and packaging, as well as flexibility in usage, in contrast -to the freeze-dried ~ ~, or dry media preparations.
The liquid media which is designed to provide for stabilization o~ enzymes and coenzymes as hereinafter described is uniquely formulated so that one or more coenzymes may be stabilized in the media. Otherwise, one or more enzymes may be stabilized in the liquid media. Moreover, both coenzymes and enzymes mayb e stabilized in the same liquid media in a single container.
Stabilization of the enzymes and/or coenzymes is accomplished by dissolving a polymer, such as a gelatinl in distilled water. The gelatin is preferably dissolved on a 0.1% w/w basis. Thereafter, the solubilized gelatin in water is heated to about 30 to fully dissolve the gelatin. In some cases, an azide compound may be used, which not only serves as a bacteriostat, but as a stabilizer as well. Thereafter, this solution is cooled down essentially to room temperature, or about 20C.

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¦ In one case, the coenzyme, nicotinamide-adenine dinucleo-; 21 tide (NAD), is added to the solution, along with a buffering ~ I agent, such as tris(hydroxvmethyl) aminomethane, for purposes i 41 of adjusting the pH. In this case, the pH is adjusted approxi-¦ mately between about 6.0 to about 8.5 with a preferred pH of ¦ 7.5. After the addition of the coenzyme, a polyol, such as glycerol, is added on abou a 30~ v/v basis. After addition of ¦ the polyol, the pH may again be adjusted to about 7.5.
9 I In accordance with the present invention, more than one lO¦1 coenzyme may be stabilized in the above-mentioned solution. In this case, the other of the coenzymes could be added prior to 12 li or after the addition of the NAD. For example, in one embodi- ¦
ment of the present invention, adenosin triphosphate (ATP) may l4 be added as the other coenzyme. -15 1 After the addition of the coenzymes and the adjustment l~ 1 of the pH of the liquid, an enzyme, such as hexokinase (HK), may 17 ¦ also be added. Typically, the hexokinase would be added from 18 a suspension, such as a glycerol suspension, or an ammonium l9 1 sulfate suspension. Another enzyme may also be added, as for 20 ¦ example, glucose-6-phosphate dehydrogenase.
21 1 After the liquid stabilized enzyme and/or coenzyme solution 22 i is prepared, it is then dispensed into amber-glass bottles and -~ 23 ¦ which are sealed in an air-tight condition. Moreover, these 24 1 bottles are typically stored under refrigeration. The projected shelf life of the stabilized enzymes and coenzymes is up to . 26 four years under these conditions without apprec1able degradation.
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:~ 1 DETAILED DESCRIPTION
~ 2 In the clinical diagnostic field, the commercial applicaticn t' 3 of the present inven-ti~n is represented by, but not limited to,

4 the diagnostic reagents used to determine subs-trate concentration, as for example, glucose concentrations in bioloyical fluids, and 6 the like. Nevertheless, compositions prepared in accordance with the present invention can be used to determine and quantitate 8 other bi~logical conslltuents, as for example, the following con-stituents in biological fluids:
1. Glutamic-oxalacetic transaminase (SGOT) 11 2. Glutamic-pyruvic transaminase (SGPT) 12 3. Lactic dehydrogenase (LDH-P) 13 4. Lactic dehydrogenase ~LDH-L) 14 5. Creatine Phosphokinase (CPK) 6. a-Hydroxybuteric dehydrogènase (~-HBD) 16 7. Glucose (via Hexokinase-G-6-PDH) 17 These above-identified reagents often react similarly, contain 18 some common labile ingredients, and some of the chemical reactions 19 involved are common. The following chemical reaction scheme is ~; ZO presented as a model to illustrate the general nature of the 21 reactions involved:

. _ _ 2~ Enzyme 1 s 24 (1) SUBSTRATE (S) ~ ~ PRODUCT(S) 25 -- - pH
~ 26 Enzyme 2 s 27 (2) PRODUCT/SUBSTRATE+NAD-NADH2 ~ NADH2-NAD+PRODUCT
28 ~--Catalyst 30 (3) NADH2 + CHRO~OGEN - CHROMOGEN + NAD

32 (oxidized) (reduced) .~, ~:

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1 All enzymatic reactions listed above, in accordance with 2 this invention, will ollow this general scheme, where reaction .
t2) is usually referred to as the coupling reaction, reactions (2) or (3) are the measuring reactions, and reaction ~1) may be characterized as the primary reaction. It is understood, however, that not all three reac-tions are required for measurernent;
7 in fact, they may be limited to two, or one. In the case of 8 the ultraviolet measurement of lactic dehydrogenase (LD) activity, . 9 only reaction (2) is involved, as follows:

12 LACTATE + NAD ( > NADH2 + PYRUVATE

14 Conversely, more than the three reactions listed may be involved, as in the case of Creatine phosphokinase (CPX):

CPK
18 ~1) GP * ADP C ~ ATP + CREATINE
: 19 ,~
HK
21 (2-) ATP + GLUCOSE < ~ GLUCOSE-6-PHOS. + ADP
22 .

2~ (3) GLUCOSE-6-PHOS. + NAD ~ ~ NADH2 -2~ `~-28 (4) NADH2 + INT < (red) 31 .

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1 ¦ SY~BOLS:
2 ¦ CP = Creatine phosphate 3 ! CPK = Creatine phosphaze ADP = Adenosin -5'-diphosphate

5 ¦ AM = Adenosin monophosphate ¦ ATP = Adenosin triphosphate ¦ HK = Hexokinase 8 ¦ NAD = Nicotinamide-adenine dinucleotide 9 ¦ - NADP = Nicotinamide-adenine dinucleotide phosphate 10 ¦ NADH2 = Nicotinamide-adenine dinucleotide, reduced ll ¦ GLDH = Glutamate dehydrogenase 12 ¦ G-6-PDH = ~lucose-6-phosphate dehydrogenase 13 ¦ G-6-P = Glucose-6-phosphate 14 ¦ INT = Tetrazolium salt PEP = Phosphoenol pyruvate 16 ¦ PMS = Phenazine methosulfate ¦ PK = Pyruvate kinase l9 ¦ In this case, reactions (2) and (3) may be considered the coupling 20 ¦ reactions, reactions (3) or (4) the measuring reactions, and 21 ¦ reaction (l) the primary reaction.
22 ¦ Referring to REACTION SCHEME 1 -- GENERAL MODEL, it becomes .
23 ¦ obvious and is general knowledge that the use of the reaction 24 ¦ sequence permits the analytical quantitation of either the reaction 25 ¦ substrates/products or the catalyzing enzymes.
26 ¦ The quantitation of these constituents in biological -Eluids 27 ¦ is a well accepted and widely used diagnostic tool in diagnosis 28 ¦ and treatment of human and animal disease states.
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1 ~nzymes are large molecular weight, complex protein molecules, 2 usually of unknown chemical structure. They are presently classi-3 fied by their catalytic activity and extreme substrate specificit~.
~ Enzymes may be redefine~ as biological catalys-ts, capable of catalyzing a reaction of a single substrate, or a reaction of a

6 similar group of substrates.

7 Coenzymes are lower molecular weight organic chemicals o~

8 well-defined structure, whose reactions or interactions are neces-

9 sary for specific enzyme assay or reaction. They are catalyzed resulting in a reversible change in the coenzyme's structure 11 and/or atomic composition. Coenzymes are very us~ful in clinical 12 assay procedure. Some have strong absorbance, their reactions 13 are stiochiometric with the substrate and, therefore, the creation 14 or disappearance of the absorbing form can be followed photo-metrically. Nicotinamide-adenine dinucleotide (NAD) and its 16 reduced form (NADH2) are used in many important clinical assays 17 such as the S.G.O.T., S/P.G.T. and ~DH assays previously described.
18 NAD and NADH2 have a molecular weight of about 700 and are very 19 complex organic molecules. NADH2 absorbs strongly at 340 nm, whereas NAD does not absorb at this wave length.
21 Substrates are organic chemicals of known structure, whose 22 reactions or interactions are catalyzed by enzymes resulting 23 in a change in the compound's structure, atomic composition, or 2d stereo-chemical rotation. In general, substrates are prone to ;
microbiological degradation as they serve as food for bacteria, 26 fungi, and other microorganisms. Otherwise, these compounds 27 remain stable in aqueous media at or near neutral pH (I.e., pH
28 ra~ge of 4-10). ~ypical substrates are glucose, lactate or 29 lactic acid, gluconate and the like.

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The following reactions illustrate the determination of 2 ¦ glucose by utilization of the coenzymes ATP and NAD.
3 ¦ H~
4 ¦ GLUCOSE + ATP ,~ G-6-P -~ ADP
5 l 6 ¦ G-6-P ~ NAG ~ ~ NADH + 6-PHOSPHOGLUCONIC ACID
7 l 8 ¦ The enzyme which causes the primary reaction is hexokinase, and 9 ¦ the enzyme which causes the coupling and measuring reaction is

10 ¦ G-6-PDH. In the above reaction, the glucose is dete`rmined by li ¦ measuring the NADH which is formed in the measuring reaction.
12 ¦ In essence, the reaction is permitted to go to completion, and 13 I the amount of the coenzyme NADH formed is essentially measured.
14 ¦ NAD, while being unstable in water and in dry form when 15¦ exposed to humid environments, is not nearly as unstable as the 161 reduced from NADH2. Accordingly, the NADH2 must be kept free of 17 ¦ moisture, whereas the NAD may be packaged in a container with~an 18 ¦ aqueous solution, although stabilIzed in accordance with the 19¦ present invention. Stability is better in an acid pH, wher~as in an alkaline pH, there is a tendency for the NAD to decompose.

22 Neither the exact mechanism, nor the end products, are of signi-ficance, except that the decomposed NAD can no longer effectively 23 function as a coenzyme, nor does it possess the extenction coeffi-2a cient at the necessary wave length. -One of the unique advantages of the present invention is 26 that all components may be stabilized in a single reagent bottle.
27 Generally, there are two primary considerations in the formulation 28 of a stabilized enzyme or coenzyme. The first of these considera-2g tions is that of providing a highly stable enzyme or coenzyme in -10- ~

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1 a liquid media, and the second consideration is to limit the 2 number o~ packa~es as much as possible. In the stabilization of 3 coenzymes, such as NAD, it has been observed that the NAD is 4 far more stable than the NAD~. Consequently, it is not necessary to use the complex stabilization -techniq~es necessary ~or NRDH.
6 Accordingly, all reagents can be packaged in one solution.
7 In stabilizing the enzymes and coenzyrnes of the present 8 invention, a polyrner, such as a gelatin, is dissolved in distilled ~ water. The polymer is preferably present in the stabilized solu-tion up to an amount that remains in homogeneous suspension under li refrigeration without precipitation. The polymer should be 12 present in an amount from about 0.01% to about 0.5~ based on the 13 total composition, and preferably within a range of 0.05~ to 1~ about 0.25%. Any water-soluble polymers which are useful as stabilizing agents in this invention are those which do not 16 inhibit enzymatlc activity and are capable of entrapping the 17 enzyme in the polymer matrix. The polymer may be a synthetic 18 or organic material, such as polyvinylpyrrolidine or dextran of 19 biologic orgin, such as gelatin which is denatured collagen.
The polymer may be dissolved in the water by heating, 21 generally to above 30C. The rate in which the polymer is dis-22 solved will increase with an increase in temperature.
23 After the polymer has been completely dissolved in water, 2a an azide compound, such as sodium azide, may be added, preferably in amount o about 0.1% w/w. However, the amount of azide com-26 pound which is added can range from 0.01~ to about 0.5~. It .
27 has been found in accordance with the present invention that the 2~ azide compound exhibits the rather surprising result of aiding 29 in the stability of the enzymes. Previously, it was only thought ,. , , ' .lZ ~

1 ~ that t azide compound served as a bacteriostatic agent or 2 bactericide. Nevertheless, while the complete mechanism of 3 stabilization with the azide compound, in combination with the other ingredlents, is not fully understood, it has been estab-lished that the azide compound does, nevertheless, provide 6 increased stability. In many cases, the azide salt is not neces-7 sary and can be eliminated. Thus, in many cases, the polymer 8 and organic solvent in the aqueous media are sufficient to pro-9 vide the required stabilization of the labile components. In some few cases, the azide salt must be eliminated inasmuch as

11 it may have a tendency to interfere with stabilization, or other-

12 j wise materially affect a substrate, as for example, glucose.
~3 In addition to the foregoing, other bactericidal or other lg fungicidal agents which do not chemically react with a substrate 15 I or inhibit the enzymatic reaction may be employed. For example, 16 I some of these agents which may be used in addition to sodium 17 ~ azide are benzoic acid, phenol, thymol or pentachlorophenol.
18 ¦ In some cases/ it may be desirable to employ a metal, such 19 I as magnesium, ~hich aids in initiation of a reaction when the 20 I stabilized composition is used. Magnesium, in the salt form 21 j of magnesium chloride is one of the preferred agents for this 22 I purpose. This agent does not have to be incorporated in the 1 6 23 ! stabilized compositions of the present invention and may be 24 ! added at the time of use. This agent which activates the coupling 25 ¦ enzymes should be used in an amount of about 0.01% to about 1%
26 and preferably about-0.03%.
I At this point in the process, the solution may be cooled 28 ¦ to about room temperature, such as about 20C to about 25C in 29 a wate hath. After the solution has be-n cooled, a buffering .

S~ , 1 ¦ agent, such as tris(hydroxymethyl) aminornethane may be added.
2 ¦ Typically, this buffering agent is added in an amount of about 3 ¦ 50 millimoles to about 200 millimoles, but at least sufficient to ¦ maintain the pH within a range of 6.0 -to about 8.5. Other known ~ ¦ buffering a~ents and other forms of buffering agents may also be `; 6 I employed in the process. In some cases, buffer salts of the type ¦ hereinafter described may be used. The buffer salt is added in an 8 ¦ amount necessary to maintain the pH between 6~0 to 8.5. 6enerally, 9 ¦ the buffer is a combination of .1 - 1~ of an alkali metal hydroxide 10 ¦ and 0.5 to 3% of an alkali metal acid carbonate or phosphate.
11 ¦ The total salt content also effects the amount of polymer required.
12 ¦ At higher salt content, e.g. above 4% by weight, less polymer

13 ¦ is required due to the electrostatic stabilization provided by

14 ¦ the salt. However, at higher salt content, the polymer may

15 ¦ cloud the solution or precipita~e requirin~ warmihg the solution

16 ¦ to redissolve.

17 ¦- - After the pH of the solution has been adjusted to the desired

18 ¦ range, the first of the coenzymes, such as the ATP or thè NAD,

19 ¦ etc., may be added. ~n this case, the ATP is added on a basis

20 ¦ of about 0.3 millimoles to about 30 millimoles, based on the

21 ¦ total composition.

22 ¦ As indicated previously, it is possible to form solutions ,

23 ¦ of both stabilized enzymes and coenzymes. Thus, two or more 2a ¦ coenzymes and two or more enzymes may be stabilized in the same 25 ¦ solution. For example, the coenzyme ATP may be stabilized in 26 ¦ the manner as described herein. On the other hand, the NAD may 27 ¦ also be stabilized individually in the manner as described herein.
28 ¦ Nevertheless, when stabilizing two or more coenzymes, the coen-zymes m y general`y be added simu1tane-u~ly or 1n any order. The 32 ~ -13-.

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1 ¦ NAD is preferably added in a range of about .6 millimole to about , 2 ¦ 60 millimoles, based on the total composition.
3 ¦ At this point in the process, the pH should again be adjusted ¦ to at least within the range of 6.5 to about 8.0 or less, and, 5 I preferably, to 7.5.
6 ¦ After adjus-tment of the pH, a suitable organic solvent, such ¦ as glycercol, may be added. In this case, it is added within 8 ¦ the range of 25% to 40~ v/v, although, in the most preferred ¦ aspect, 30~ v/v of the organic solvent is added. However, the 10¦ amount of organic solvent could range from about 5~ to 70% v/v.
li¦ The organic solvent shoul~ have the following characteristics:
12 ¦ 1. pH range of 4 to 10;
13 ¦ 2. Liquid at room and refrigerator temperatures;
14 ¦ 3. Does not react with NAD or ATP and the like other 15 ¦ than forming electrostatic (i.e., hydrogen) bonds;
16 ¦- 4. Miscible with water;
17 ¦ 5. Standard free energy of solvolysis is low (normal l$ 1--- resonance is established).
19 ¦ The solvent must be miscible with water, liquid at room and 20 ¦ refrigerator temperatures, and non-degradatively reactive with 21 ¦-reactive sites of the enzymes and coenzymes other than formation 22 ¦ of electrostatic bonds. Useful solvents are generally stable ~, 23 ¦ organic solvents such as ethers, ketones, sulfones, sulfoxides 2~ ¦ and alcohols such as methanol, ethanol, propanol, butanol, acetone, 25 ¦ dloxane, DMSO, dimethylsulfone and THF. However, higher activity-26 ¦ at lower solvent concentration for the treat~ent step is found 27 ¦ for liquid polyol solvents. Liquid polyols containing from 28 ¦ 2-4 hydroxyl groups and 2-10 carbon atoms are preferred, such as 30 ¦ glycero , ethylene glycol, propylene glycol or but~ne dlol.

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1 ~ Glycerol, propylene glycol, 1,2-propanediol, were found to ¦ possess all these qualities and are the solvents of choice.
3 ¦ When the selected organic solvent is a polyol, it is not * ¦ necessary to use the azide compound, or, for -that matter, oth~r 5 ¦ bacteriostatic agents, since the polyol effectively functions 6 ¦ as a bacteriostatic agent. Nevertheless, while the selected 7 ¦ solvent and the polymer provide the the required stability in ¦ an aqueous solution, the azide compound is sometimes preferable, 9 ¦ inasmuch as it appears to increase the coupling between the 10 ¦ polymer and the enzymes.
11 ¦ After the glycerol or other polyol is added, the pH of 12 ¦ the solution thus formed is readjusted. Typically, the pH may 13 ¦ be slightly basic and, therefore, a 1 normal HCl can be added 14 ¦ in order to adjust the pH. In like manner, if the pH is slightly 15 ¦ acidic, then a suitable base may be added to achieve a pH of 16 1 7.5. ~
17 ¦ One of the important aspects is that the coenzyme NAD is 18 ¦ present in excessive amounts. As indicated, the determination 19 ¦ of glucose is accomplished by measuring the NADH which is formed 20 ¦ from the NAD. The NADH is unstable in an acidic environment 21 ¦ and will degrade at a pH of 6 and, even moreso, will degrade 22 ¦ extremely rapidly at a pH of 4. The pH of the solution is there-23 ¦ fore maintained above a neutral pH of 7. While the NAD is actu-2~ ¦ ally more stable in the acid environment, it has been found in 25¦ accordance with the present invention that it does not materially 26¦ degrade in a slightly basic environment of a pH of 7.5. Never-27 ¦ theless, the NAD is added in considerable excess so that there 2~1 is always sufficient undegraded NAD present, even after several 29 ¦ years in this liquid environment.

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1 Generally, all coenzymes will be present in an amount of 2 at leas-t sufEicient to perform the desired determination. There 3 is typically no maximum amount of coenzyme present, although 4 the maximum amount will be limited by comme~cial practicalities.
After the coenzymes have been added to the liquid solution, 6 the selected enzymes may be added. As with the case of the 7 coenzymes, the enzymes may be added in any order. Again, one or 8 more enzymes may be added to the solution. In the preferred 9 aspect of the invention, and in accordance with the enzyme system identified above, the two enzymes are HK and ~-6-PDH.
11 The HK is also preferably added in no less than 111 I.U. per liter 12 ¦ (pH of 7.6, 25C). However, it is preferable to add at least f3 1,000 I.U. per liter of the HK.
1~ I The G-6-PDH should, preferably, be formed from the ?
15 ¦ L-mesenteroides bacteria, and should be concentratèd in a range lG I of about 100 I.U. per liter to about 30,000 I.U. per-Liter or -17 I above. In the preferred aspect of the invention, it is normally 18 ' about 3,000 I.U. of the G-6-PDH of this type which is used at -~ ¦
19 I a pH of about 7.8 at 25C. ~~
20 ¦ The enzymes should each be present in an amount of at 21 l` least 100 I.U. (International Units) per liter, although in 22 I most commercial reagents, the enzyme, as for example, the hexo- ¦ -23 inase, should be present at about a minimum of 1,000 I.U. per 2a liter. In the normal commercial packages, the enzyme is present- ¦
in about 1,000 to about 10,000 I.U. at a pH of 7.6 and at a 26 temperature of ~' 1.

~.~2~V55 1 about 25C. However, the ma~imum amount of the enzyme is 2 unlimited, although, normally, in almost all applications the 3 amount of enzyme will not exceed 100,000 I.U.
It is important in -the process of the present invention 5 that the enzymes are added after the final pH is adjusted.
6 While the full mechanism for accomplishing the stabili~ation 7 of the enzymes and coenzymes is not fully understood, it is 8 believed that the selected solvent stabilizes the enzyme in the 9 liquid media by protecting the ~unctional group site, that is the part of the molecule where a substrate reaction may actually 11 occur, or is otherwise catalyzed. Moreover, stabilization is 12 believed to occur by protecting the enzymes and coenzymes from 1~ microbial contamination and thus degradation. The coenzyme NAD
1~ differs from the coenzyme NADH in that the NAD will not appreci-ably dissolve in the selected solvent, such as propyleneglycol.
16 However, the NAD iS more stable in water and the coenzyme does 17 appear to be stabilized by the polyol. A pure polyol will ¦ -18 l denature the enzymes, but in the presence of an aqueous solution, 19 I such as a water-solvent mixture, the enzymes do not denature.
20 ¦ Apparently, a polar ~roup is required in the organic solvent to 21 I maintain the active sites of the enzymes in a stable condition.
22 I Obviously, some form of physical or chemical reaction occurs in 23 the concentrated aqueous-organic solvent media, inasmuch as the

24 enzymes and coenzymes retain catalytic activity and do not

25 I de~rade in the specified concentrations.

26 In addition to the above, the polymer appears to react in

27 some fashion with the azide compound in order to form an electro-

28 static or covalent bond between the enzymes and the polymer. In

29 essence, it may also appear that the polymer may stretch to some-what encapsulate, and thereby protect, the active sites of the 31 enzymes. In this way, enzyme dénaturation or other form of 32 degradation is inhibited or does not occur~

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1 As indicated above it is possible to stabilize at 2 least t~o or more encoenzymes or at least two or more enzymes in the same solution. Moreover, and more importantly, it is 4 possible ~o stabilize both en~ymes and coenzymes in the same solution. It is believed that the aqueous solution of the 6 organic solvent is the primary factor in stabilizing the 7 coenzyme although the polymer does appear to provide some 8 stabilizing effect. In stabilization of the enzyme, the 9 organic solvent and the polymer appear to be the primary factors resulting in stabilization. In addition, and in many 11 cases the a~idesalt aids in increasing stabilization. In 12 either case, it can be observed that both enzymes and coen-13 zymes are still in the same single solution.
14 Some of the additional reactions which have been performed with the stabilized enzyme and coenzyme compositions 16 are set forth below. The reaction involving the phosphory-17 lation of creatine is:
18 CK >
lg -CREATI~IP + ATP CP + ADP
pH 8-9 22 The remaining reactions are all self explanatory with reference 23 to the list of symbols set forth above. For an NADP reaction, 25 G-6-P + NADP ~ NADPH + 6-phosphogluconic acid 26 <

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2I For an P react on, 3 CP + ADP ~ CREATINE -~ ATP
4 pll 6-7 6 For the following reaction the starting reaction of creatine +

8 ATP would be employed to provide the ADP, Thereafter, 9 ADP + PEP ~ ATP ~ Pyruvate ' 11 ~ ~

12 Pyruvate + NADH ~ > Lactate + NAD

14 The following reactions show the use of urease and GLDH enzymes 15 in the stabilized liquid compositions.

UREASE

18 UREA ~ 2~1H4 + C02 rI~4 + ~-Ketoglutarate + ~IAD ~ ~lutamate + IIADH
21 The invention is further illustrated by, but not 23 limited to, the following Examples:

24 About 0.7 grams of a gelatin polymer is added to about 700 milliliters of water. This solution is then heated above 27 30C in order to dissolve the gelatin polymer.

28 After the addition of the polymer the solution is in-29 serted in a water bath in order to reduce the temperature to 31 about 22C.

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l¦ The p~l is then adjusted within the range of ~.5 to 21 8Ø ~fter the temperature has been reduced and p~l adjusted, 31 ahout 2.0 grams of ATP is added to the solution, which is in 41 turn followed hy 4.0 grams of I~AD. Three grams of a ma~nesium 51 chloride salt is added along with the ~lAD. Three hundred ml 61 Glycerol is then added.
71 After the addition of the glycerol, the p~l is adjusted ~¦ to about 7~5 by the addition of l normal hydrochloric acid.
9¦ After complete solution is attained, the solution is lO¦ added to a plastic or glass container, which is then closed.
ll ¦ The containers are sealed and stored under refrigeration. It 12 1 has been found that a stabilized coenz~e composition in this 13 ¦ manner provides a storage stability of up to four years with-14 ¦ out significant degradation.

16 ¦ II
17 ¦ The sample produced in accordance with ~xample I is 18 ¦ provided with the enzyme hexol~inase prior to sealing in the l~ ¦ glass contalnerO The same shelf life is obtained without 20 ¦- significant degradation.

23 ¦ The sample of Example II is also provided with the 241 enzyme G-6-PDH prior to sealing in the glass container and the 251 same long shelf-life without significant degradation is obtained.
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1 bout 1.0 grams of a dextran polymer is added to about 3 700 milliliters of water. This solution is then heated above 4 30C in order to dissolve the polymer. The solution is in-serted in a water bath in order to reduce the temperature to 6 about 22C.
7 After the temperature has been reduced, about 2.2 8 grams of NADP iS added to the solution, which is in turn 9 followed by 4.0 grams of ATP. The pH of the solution is adjusted within the range of 6D 5 to 8.0, 325 ml gl~cerol is 11 then added.
12 After the addition of the glycerol, the pH is adjusted 13 to about 7.5 by the addition of 1 normal hydrochloric acidO
14 After complete solution is attained, the solution is added to a plastic or glass container, which is then closed.
16 The containers are sealed in an air-tight manner and stored ;' 17 under refrigeration. It has been found that a stabliized 18 coenzyme composition is about as effective as the composition of Exampie I, even though the azide salt was not added.
21 `
22 The following examples are set forth in schematic form 23 but show the reagents and the amounts added to the various 24 important steps in producing the stabilized compositions of the present invention.

.,1~
~ 5~ , 21 Stabiliæed ADP ? AMP and NAD
3¦ About 700 ml of water 41 0.5 grams of gelatin dissolve with lleat 0.7 grams of sodium azide 71 cool to room temperature ~¦ 50 grams of creatine phosphate 9 4 grams of ADP
10 ¦ 20 grams of AMP
11 ¦ 15 grams of IIAD
12 ¦ dissolve and adjust pll between 7 to 9 13 300 ml glycerol 14 mix and readjust pH
Package in a bottle and seal.

18 Stabilized ADP, AMP, NAD, H~ and G- 6-PDH I
19 ¦ About 300 I.U. to about 15,000 I.U. per liter of 20 ¦ G-6-PDH and about 100 I ~Uo to about 10,000 I.U~ per liter of 21 ¦ HK is added to the solution of E~ample V prior to paclcaging 22 ¦ thereof.
1~ 231 2~¦ VII
251 1.5 grams of NAD
26¦ Dissolve in 5 ml water 271 Add 5 ml of glycerol : Adjust pH to less than 5 2~

31 .

7~5~ .

l VII
2 Stabilization of ~IAD and ~l}~ I
____ _ 3 1.5 grams oE NAD
4 Dissolve in 10 ml of p~l 7 h~ Fer of 0.1 molar P~PES*
buffer 6 Adjust pH to 6 to 7 :~ 7 Add 10 ml. of glycerol ~ 8 ~eadjust pH
: 9 Add and dissolve 1~ mg. ~IK of activity of 150 I.U.
per miligram.

12 *PIPES = PIPERAZIrlE [BIS] E,THANE SULFONIC ACID
1;3 , IX
16 Stabilization of creatine, ATP and PEP
17 1, oon ml of water 18 Add 12.1 grams of tris(hydroxymethyl) aminomethane 19 Add 1.0 ~rams gelatin Dissolve with heat above 30C
21 Cool to room temperature 22 Add 2.0 grams ATP
23 Add 2 grams PEP
24 . Add 10.0 grams creatine 26 issolve and adjust pd to 9 2~

31 . .

Il,, ~7~5 1 ~ X
2 To the stabilized solution of Example IX, 3 100 I.U./liter -to 10,000 I.U./liter of J.VH was added and ~; 100 I.U./liter to 10,000 I.U./liter of PK was added, 6 prior to packagin~.

Each of the compositions of Examples V through X have 9 the same long shelf life without any substantial degrada~ion.
Moreover, each of the above examples are based on samples 11 I actually prepared and tested in accordance with the present 12 ' invention.
~ l i l~L l l ~1 ~ ~
16 l i 17 l' .

21 . I
22 I 1.
23 .
24 i 31 , .

,1 -24-1l .. . .

Claims (9)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A stabilized liquid enzyme composition used in biological determinations and which enzyme is unstable in an aqueous media, said composition comprising:
(a) an aqueous vehicle, (b) at least 100 I.U. of enzyme dissolved in said aqueous vehicle, (c) a non-reactive aqueous miscible organic solvent in said aqueous vehicle and which is liquid at least at room temperature, (d) a water soluble polymer which does not sub-stantially inhibit enzymatic activity, and (e) a bacteriostat which provides stabilization as well as providing bacteriostatic action, (f) said enzyme being selected from the class consisting of glucose-6-phosphate dehydrogenase, hexokinase, glutamate dehydrogenase, creatine phosphokinase, pyruvate kinase and alkali phosphatase.

2. The stabilized liquid enzyme composition of claim 1 further characterized in that said composition comprises a labile second enzyme which is also stabilized by at least said organic solvent or said polymer.

3. The stabilized liquid enzyme composition of claim 1 further characterized in that the bacteriostat is an azide compound.

4. The stabilized liquid enzyme composition of claim 1 further characterized in that said organic solvent has the following characteristics:
(a) pH between 4 to 10;
(b) liquid at room and refrigerator temperatures, (c) does not react with enzymes other than forming electrostatic (i.e., hydrogen) bonds;
(d) miscible with water;
(e) standard free energy of solvolysis is low (normal resonance is established).

5. The stabilized liquid enzyme composition of claim 4 further characterized in that said organic solvent is non-reactive with said enzyme and aqueous vehicle at room and refrigerator temperatures.

6. The stabilized liquid enzyme composition of claim 1 further characterized in that the enzyme is selected from the class consisting of G-6-PDH, HK, GLDH, CK, PK and PEP.

7. A method of stabilizing a labile enzyme used in biological diagnostic determinations and which enzyme is normally unstable in an aqueous media, said method comprising:
(a) contacting water with an aqueous miscible organic solvent to form a solution thereof and which organic solvent is liquid at least at room temperature, (b) adding at least 0.01% of a water soluble polymer to said solution, (c) adding a bacteriostatic agent which also func-tions as an enzyme stabilizing agent to said solution, (d) dissolving at least 100 I.U. per liter of enzyme to said solution to form the composition, and which enzyme cooperates in a determination reaction, (e) said enzyme being selected from the class con-sisting of glucose-6-phosphate dehydrogenase, hexokinase, glutamate dehydrogenase, creatine phosphokinase, pyruvate kinase and alkali phosphatase, (f) and sealing the composition.

8. The method of claim 7 further characterized in that said bacteriostatic agent is an azide compound.

9. The method of claim 7 further characterized in that said method also comprises adding a second enzyme to said solution which is also stabilized therein.