Note: Descriptions are shown in the official language in which they were submitted.
1 BACKGROUNI) OF THE INVENTION
2 I. Field of the Invention
3 This invention relates in general to certain new and
useful improvements in the stabilization of enzymes 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 sta-te of the art used for stabilizing
}1 the reactive ability of enzymes or coenzymes is by locking them
12 into a solid matrix, either by freeze drying, dry blending such
13 as used for tableting dried powders, primarily in the pharmaceu-
1 a tical diagnostic and related industries and immobilization by
locking the chemlcal structure of the enzyme into a solid matrix.
16 ¦ Contrary to the sophistication these terms imply, these approaches
17 I 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 drylng and dry blending, and usefulness of the produce
2~ I is further limited by packaging modes and sizes.
24 ¦ Furthermore, yood 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
28 I + 10~ of the mean.
29
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1 OBJECTS OF THE INVENTION
2 It is, therefore, the primary object of the present inven-
3 tion to provide a liquid composition with coenzymes and/or
4 enzymes which are stabilized in a single container.
It is a further object of the present invention to provide
6 a labile enzyme and coenzyme composition of the type stated in
7 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.
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 o-therwise other
73 labile enzymes and which composition has a long shelf life.
1 . I
16 l l
17 I i
1~3 I .
1~ i
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22
23
24
27
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1 S MMARY OF THE INVENTION
2 Labile enzymes and coenzymes are treated according to the
3 invention, resulting in long-term stabili~y wlthout affecting
4 enzymatic or coenzymatic reactivity or phometric absorptivity.
Providing enzyme and coenzyme reagents in a stable liquid form
enhances the colorime-tric applicability o~ present ~ay NAD/N~DT~
7 coupled methodologies, as well as other methodologies~ primarily
8 because the separation of ingredients is easily accomplished.
9 Stable li~uid reagents are especially advantageous where NADH
and other coenzyme consumption is the basis of measurement and
11 the color reagent must be separated from NADH and the reaction
12 main. In the ultraviolet mode, the li~uid system offers be-tter
1~l reagent homogeneity and packaging, as well as flexibility in
1'~ ' usage, in contrast to the freeze-dried or dry media preparations. I
15 ¦ The liquid media which is designed to provide for stabilizattc
16 ¦ of enzymes and coenzymes as hereinafter described is uniquely
17 I formulated so that one or more coenzymes may be stabilized in
18 , the media. Otherwise, one or more enzymes may be stabilized in
19 the liquid media. Moreover, both coenzymes and enzymes may be
20 I stabilized in the same liquid media in a single container.
21 ¦ Stabilization of the enzymes and/or coenzymes is accom~ I
22 , plished by dissolving a polymer, such as a gelatin, in distilled li
23 j water. The gelatin is preferably dissolved on a 0.1% w/w basis.
2~ ¦ Thereafter, the solubilized gelatin in water is heated to about
25 1 30 to fully dissolve the gelatin. In some cases, an azide
26 compound may be usedl which not only serves as a bacteriostat,
27 but as a stabilizer as well. Thereafter, this solution is
2~ cooled down essentially to room temperature, or about 20C.
29
32
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1 In one case, the coenzyme, nicotinamide~adenine dinucleo-
2 tide (NAD), is added to the solution, along with a buffering
agent, such as tris(hydroxy~ethyl) aminomethane, for purposes
4 of adjusting the pH. In this case, the pH is adjusted approxi-
~ately between about 6.0 to about 8.5 with a preferred pH of
6 7.5. After the addition of the coenzyme, a polyol, such as
7 glycerol, is added on abou a 30% v/v basis. After addition of
8 the polyol, the pH may again be adjusted to about 7.5.
9 I In accordance with the present invention, more than one
10 I coenzyme may be stabilized in the above-mentioned solution. In
11 l this case, the other of the ~oenzymes could be added prior to
12 ¦ or after the addition of the NAD. For example, in one embodi-
13 ' ment of the present invention, adenosin triphosphate (ATP) may
1~ ~ be added as the other coenzyme.
After the addition of the coenzymes and the adjustment
16 of the pH of the liquid, an enzyme, such as hexokinase (HK), may
17 ¦ also be added. Typically, the hexokinase would be added from
~8 1 a suspension, such as a glycerol suspension, or an ammonium
19 ~ sulfate suspension. Another enzyme may also be added, as for
20 I example, glucose-6-phosphate dehydrogenase.
21 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
2~ 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 appreciable degradation.
27
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1 D TAILED DESCRIPTION
2 In the clinical diagnostic field, the commercial application
3 of the present invention is represented by, but not limited to,
4 the diagnostic reagents used to determine substrate concentration,
as for example, glucose concentrations in biological fluids, and
6 the like. Nevertheless, compositions prepared in accordance
7 with the present invention can be used to determine and quantitate
8 other biological cons~-tuents, as for example, the following con-
9 stituents in biological fluids:
1. Glutamic~oxalacetic transaminase ~SGOT)
1l 2. Glutamic-pyruvic transaminase (SGPT)
12 3. Lactic dehydrogenase (LDH-P)
13 4. Lactic dehydrogenase (LDH-L)
14 5. Creatine Phosphokinase ~CPK)
6. ~-Hydroxybuteric dehydrogenase (~-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
l9 involved are common. The following chemical reaction scheme is
presented as a model to illustrate the general nature of the
21 reactions involved:
22 REACTION SCHEME l ~- GENERAL MODEL
2~ _ _ _
Enzyme l
24 (1) SUBSTRATE (S) ~_ ~ PRODUCT(S)
pH
26 Enzyrne 2
27 (2) PRODUCT/SUBSTRATE+NAD-NADH2 ~ ~ NADH2-NAD+PRODUCT
28 pH
~9
Catalyst
(3) NADR + CHROMOGEN = CHR0~10GEN + NAD
3~ 2 ~oxidized) (reduced)
-6~
1102~5
1 A11 enzymatic reactions listed above, in accordance with
2 this invention, will follow this general scheme, where reaction
3 (2) is usually referred to as the coupling reaction, reactions
4 (2) or (3) are the measuring reactions, and reaction (1) may be
characterized as the primary reaction. It is understood, however,
6 that not all three reactions are required for measurement;
7 in fact, they may be limited to two, or one. In the case of .
8 the ultraviolet measurement of lactic dehydrogenase (LD) activity,
only reaction (2) is involved, as follows:
REACTION SCHEME 2 -- LDH
li LDH .
12 LACTATE + NAD ~ ~ NADH2 + PYRUVATE
13
14 Conversely, more than the three reactions listed may be
involved, as in the case of Creatine phosphokinase (CPK):
16 REACTION SCHEME 3 -- CPK
CPK
18 (1) GP + ADP C ~ ATP + CREATINE
19
HK
21 (2) ATP + GLUCOSE < > GLUCOSE-6-PHOS. + ADP
22
23
G-6-PDH
2~ (3) GLUCOSE-6-PHOS. + NAD ~_ _ ~ NADH2
26
PMS
27 (4) NADH2 + INT ~ ~ INT + NAD
28 (ox) (.red)
29
33l
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~7-
2~
1 SYMBOLS:
2 CP = Creatine phosphate
3 CPK = Creatine phosphaze
4 ADP = ~denosin -5'-diphosphate
AM = Adenosin monophosphate
6 ATP = Adenosin tri.phosphate
7 HK = Hexokinase
8 NAD = Nicotinamide-adenine dinucleotide
9 NADP = Nicotinamide-adenine dinucleotide phosphate
NADH2 = Nicotinamide-adenine dinucleotide, reduced
11 GLDH = Glutamate dehydrogenase
12 G-6-PDH = Glucose-6-phosphate dehydrogenase
13 G-6-P = Glucose-6-phosphate
14 INT = Tetrazolium salt
PEP = Phosphoenol pyruvate
16 PMS = Phenazine methosulate
17 PK = Pyruvate kinase
18
19 In this case, reactions (2) and (3) may be considered the coupling
reactions, reactions (3) or (4) the measuring reactions, and
21 reaction (1) 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 reacti.on
substrates/products or the catalyzing enzymes.
26 The quantitation of these constituents in biological fluids
27 is a well accepted and widely used diagnostic tool in diagnosis
28 and treatment of human and animal disease states.
. 29
31 ~
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1 Enzymes 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 specificity.
4 Enzymes may be redefined as bioloyical catalysts, 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 of
8 well-defined structure, whose reactions or interactions are neces-
9 sary for specific enzyme assay or reaction. They are catalyzed
resulting in ~ reversible change in the coenzyme's structure
li and/or atomic composition. Coenzymes are very useful in clinical
12 assay procedure. Some have strong absorbance, their reactions
13 are stiochiometric with the substrate and, therefore, the creation
1~ or disappearance of the absorbing form can be fol]owed 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 LDH assays previously described.
18 NAD and NAD~2 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
2a stereo-chemical rota-tionO In general, substrates are prone to
Z5 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 range of 4-10). Typical substrates are glucose, lactate or
29 ~ lactic acid luconate and the 11]ce.
32 _9_
ll~Z~:Z~i
l ¦ The following reactions illustrate the determination of
21 glucose by utilization of the coenzymes ATP and NAD.
31 HK
GLUCOSE + ~TP ~ G-6~P + ADP
l G-6-P~H
61 G-6-P + NAD < ~ NADH ~ 6-PHOSPHOGLUCONIC ACID
71
81 The enzyme which causes the primary reaction is hexokinase, and
91 the enzyme which causes the coupling and measuring reaction is
0¦ G-6-PDH. In the above reaction, the glucose is determined by
ll¦ measuring the NADH which is formed in the measuring reaction.
12¦ In essence, the reaction is permitted to go to completion, and
13 ¦ the amount of the coenzyme NADH formed is essentially measured.
141 NAD, while being unstable in water and in dry form when
15 ¦ exposed to humid environments, is not nearly as unstable as the
16 ¦ reduced from NADH2. Accordingly, the NADH2 must be kept free of
17 1 moisture, whereas the NAD may be packaged in a container with an
18 ¦ aqueous solution, although stabilized in accordance with the
l9 ¦present invention. Stability is better in an acid pH, whereas
20 ¦ in an alkaline pH, there is a tendency for the NAD to decompose.
21 ¦ Neither the exact mechanism, nor the end products, are of signi-
22 I ficance, except that the decomposed NAD can no longer effectively
23 ¦function as a coenzyme, nor does it possess the extenction coeffi-
24 ¦cient at the necessary wave length~
25 1 One of the unique advantages of the present invention is
26 ¦that all components may be stabilized in a single reagent bo-ttle.
27 1 Generally, there are two primary considerations in the formulation
28 ¦of a stabilized enzyme or coenzyme. The first of these considera-
29 ¦tions is that of providing a highly stable enzyme or coenzyme in
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1 a liquid media, and the second consideration is to limit the
2 number of packages 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 NADH. Consequently, it is not necessary
6 to use the complex stabilization techniques necessary for NADH.
6 Accordingly, all reagents can be packaged in one solution.
7 In stabilizing the enzymes and coenzymes o~ the present
8 invention, a polymer, such as a gelatin, is dissolved in distilled
9 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
1~ 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 enzymatic activity and are capable o~ 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,
2~ an azide compound, such as sodium azide, may be added, preferably
25 in amount of about 0.1% w/w. However, the amount of azide com- .
26 pound which is added can ranye from 0.01% to about 0.5~. It
27 has been found in accordance with the present invention that -the
28 azide compound exhibits the rather surprising result of aidin~
29 in the stability of the enzymes. Previously, it was only thought
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1 that the azide compound served as a bacteriostatic agent or
2 bactericide. Nevertheless, while the complete mechanism of
~ stabilization with the azide compound, in combination with the
4 other ingredients, is not fully understood, it has been estab-
lished that the azide compound does, nevertheless, provide
6 increased stability. In many cases, the azide sa]t 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 p-o-
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 wise materially affect a substrate, as for example, glucose.
13 In addition to the foregoing, other bactericidal or other
1~- fungicidal agents which do not chemically react with a substrate
or inhibit the enzymatic reaction may be employed. For example,
1~ 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 as magnesium, which aids in initiation of a reaction when the
stabilized composition is used. ~agnesium, in the salt form
21 of magnesium chloride is one of the preferred agents for this
2~ purpose. This agent does not have to be incorporated in the
23 stabilized compositions of the present invention and may be
24 added at the time of use. This agent which activates the coupling
enzyrnes should be used in an amount of about 0.0]% to about 1%
26 and preferably about 0.03~.
At this point in the process, the solut;on may be cooled
28 to about room ternperature, such as about 20C to about 25C in
29 a water bath. After the solution has been cooled, a buffering
31
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1 agent, such as tris(hydroxymethyl) aminomethane 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
S buffering agents and other forms of buffering agents may also be
6 employed in the process. In some cases, buf~er salts of the -type
7 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. Generally,
9 the buffer is a combination of .1 - 1% of an alkali metal hydroxide
and 0.5 to 3~ of an alkali metal acid carbonate or phosphate.
11 The total salt content also efIects the amount of polymer xequired.
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
cloud the solution or precipita~e requirin~ warming 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 the NAD,
19 etc., may be added. In this case, the ATP is added on a basis
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
24 coenzymes and two or more enzymes may be stabilized in the same
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-
29 zymes may ge ally ~e added simulta~eously or in any order. The
32
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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 ~t this point in the process, the pH should again be adjusted
4 to at least within the range of 6.5 to about 8.0 or less, and,
6 preferably, to 7.5.
6 After adjustment of the pH, a suitable organic solvent, such
7 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
9 aspect, 30% v/v of the organic solvent is added. However, the
amount of organic solvent could range from about 5~ to 70% v/v.
11 The organic solvent should have the following characteristics:
12 1. pH range of ~ to 10;
13 2. Liquid at room and refrigerator temperatures;
14 3. Does not react with NAD or ATP and the like other
than forming electrostatic (i.e., hydrogen) bonds;
16 4. Miscible with water;
17 5. Standard free energy of solvolysis is low ~normal
18 resonance is established).
19 The solvent must be miscible with water, liquid at room and
refrigerator ~temperatures, and non-degradatively reactive with
21 reactive sites of the enzymes and coenzymes other than formation
22 o~ 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,
dioxane, DMS0, dimethylsulfone and THF. However, h~gher activity
26 at lower solvent concentration for the treatment step is found
27 for liquld polyol solvents. Liquid polyols c~ntaining from
28 2-4 hydroxyl groups and 2-10 carbon atoms are preferred, such as
glycerol, ethylene glycol, propylene glycol or butane diol.
31
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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
4 necessary to use the azide compound, or, for that matter, other
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
8 an aqueous solution, the azide compound is sometimes preferable,
9 inasmu~h as it appears to increase the coupling between the
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
1~ in order to adjust ~he pH. In like manner, if the pH is slightly
acldic, then a suitable base may be added to achieve a p~ of
16 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
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-
24 ally more stable in the acid environment, it has been found in
~5 accordance with the present invention that it does not materially
26 degrade in a slightly basic environment of a pH of 7.5. ~lever-
27 theless, the NAD is added in considerable excess so that there
28 is always sufficient undegraded NAD presen-t, even after several
29 years this liguid environment.
32 -15-
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l Generally, all coenzymes will be present in an amount of
2 at least sufficient to perform the desired determination. There
3 is typically no maximum amount of coenzyme present, although
4 the maximum amount will be limited by commercial 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
4 aspect of the invention, and in accordance with the enzyme
system identified above, the two enzymes are HK and ~-6-PDH.
ll 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
3 l,000 I.U. per liter of the HK.
l~ I The G-6-PDH should, preferably, be formed from the
L-mesenteroides bacteria, and should be concentrated in a range
16 of about 100 I.U. per liter to about 30,000 I.U. per llter or
17 above. In the preferred aspect of the invention, it is normally
18 , about 3,C00 I.U. of the G-6-PDH of this type which is-used at
l9 I a pH of about 7.8 at 25C.
20 ¦ The enzymes should each be present in an amount of at
21 ¦ least lO0 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
2~ ¦ liter. In the normal commercial packages, the enzyme is presen
25 ¦ in about l,000 to about lO,000 I.U. at a pH of 7.6 and at a
26 ~ t-mperature of
28
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29
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1 about 25C. However, the maximum 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.
4 It is important in the process of the present invention
that the enz~mes are added after the final pH is adjusted.
6 While the full mechanism for accomplishing the stabilization
7 o~ 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 functional group site, that is
lo 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 denature the enzymes, but in the presence of an aqueous solution, ¦
9 such as a water-solvent mixture, the enzymes do not denature.
Apparently, a polar group is required in the organic solvent to
21 maintain the active sites of the enzymes in a stable condition.
22 Obviously, some form of physical or chemical reaction occurs in
23 the concentrated aqueous-organic solvent media, inasmuch as the
2~ enzymes and coenzymes retain catalytic activity and do not
degrade in the speci~ied concentra-tions.
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 po]ymer. In
29 essence r it may also appear that the polymer may stretch to some-
what encapsulate, and thereby protect, the active sltes of the
31 enzymes. In this way, enzyme dénaturation or other form of
32 degradation is inhibited or does not occur. I
_ _
3LlVZ2Z5
1 ¦ As indicated above it is possible to stabilize at
2 ¦ least two or more encoenzymes or at least two or more enzymes
3 ¦ in the same solution. Moreover, and more importantly, it is
¦ possible to stabilize both enzymes and coenzymes in the same
5 ¦ 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 enz~ne, the
g organic solvent and the polyrner appear to be the primary
factors resulting in stabilization. In addition, and in many
11 cases the azidesalt 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
19 CREATINE + ATP ~ CP + ADP
C
pH 8-9
21
22 The remaining reactions are all self explanatory with reference
23 to the list of symbols set forth above, For an NA~P reaction,
24 G-6-PDH
G-6-P + NADP ~ NADPH + 6~phosphogluconic acid
26 C
27
228
31
32
~ 2 ~ Z 5
l For an ADP reaction,
2 CK
3 CP + ADP ~ CREATINE ~ ATP
pl~ 6-7
6 For the following reaction the starting reaction of creatine
7 ATP would be employed to provide the ADP. Thereafter,
8 PK
lO ADP + PEP ~ ATP + Pyruvate
~1
12 Pyruvate + NADH ~ > Lactate + ~IAD
13
14 The following reactions show the use of urease and GLDH enzymes
in the stabilized liquid compositions.
UREASE
18 UREA ~ _ 2~1 4 2
-GLDH
l9 ~ 4 + a-Ketoglutarate -t IIAD ~ ~71utamate + ~IADH
21 The invention is further illustrated by, but not
22 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
26 30C in order to dissolve the gelatin polymer.
27 -
2~ After the addition of the polymer the solution is in-
29 serted in a water bath in order to reduce the temperature to
about 22C.
~1
32 -l9-
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Z~Z5
1 The pll is then adjus~ed within the range of ~.5 to
2 8Ø A~ter the temperature has been reduced and p~ adjusted,
ahout 2.0 grams of ATP is added to the solution, which is in
4 turn followed by 4.0 grams of ~IAD. Three grams of a magnesium
chloride salt is added along with the ~IAD. Three hundred ml
6 Glycerol is then added.
7 After the additlon of the glycerol, the pll is adjusted
8 to about 7.5 by the addition of 1 normal hydrochloric acid.
9 After complete solution is attained, the solution is
added to a plastic or glass container, which is then cl~sed.
~1 The containers are sealed and stored under refrigeration. It
12 has been found that a stabilized coenz~ne 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 hexokinase prior to sealing in the
19 glass containerO The same shelf life is obtained without
significant degradation.
21
22 III
23 The sample of r,xample II is also provided with the
24 enzyme G-6-PDH prior to sealing in the glass container and the
samo lon~ s f life without significant de~radation is obtained.
29
--:ZO--
()Z;Z~5
1 IV
2 About 1.0 grams of a dextran polymer is added to about
~ 700 milliliters of water. This solution is then heated above
4 30~C 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.
q After the temperature has been reduced, about 2.2
81 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
10¦ adjusted within the range of 6.5 to 8Ø 325 ml glycerol is
~1¦ 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 acid.
14 ¦ After complete solution is attained, the solution is
15 ¦ added to a plastic or glass container, which is then closed.
16 ¦ The containers are sealed in an air-tight manner and stored
1~ ¦ under refrigeration~ It has been found that a stabliized
18 ¦ coenzyme composition is about as effective as the composition
19 ¦ of ~xampie I, even though the azide salt was not added.
21 I
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
25 ¦ the present invention.
, ~6
27
28
31 ~
I -21
, , I
Lozzzs
2 Stabilized ADP, AMP and NAD
3 About 700 ml of water
4 0~5 grams of gelatin
dissolve with heat
6 0.7 grams of sodium azide
7 cool to room temperature
8 50 gr~ms of creatine Dhosphate
9 4 grams of ADP
20 grams of AMP
~l 15 grams of IIAD
12 dissolve and adjust pH between 7 to 9
13 300 ml glycerol
14 mix and readjust pH
Package in a bottle and seal.
1~
17 VI
18 Stabilized ADP, AMP, NAD, HR and G-6-PDH
l9 About 300 I.U. to about 15,000 I.U. per liter of
G-6-PDH and about 100 I.U. to about 10~000 I.U. per liter of
21 HK is added to the solution of E~ample V prior to packaging
22 thereof.
23
24 VII .
1.5 grams of NAD
26 Dissolve in 5 ml water
27 Add 5 ml of glycerol
2B Adjust pH to less than 5
31
32 -22-
~z~
1 VII
2 Stabilization of ~AD and ~IY
3 ` 1.5 grams of NAD
4 Dissolve in 10 ml of pH 7 huffer of 0.1 molar PIPES*
buffer
6 Adjust pl~ to 6 to 7
Add 10 ml~ of glycerol
8 Readjust ~H
9 Add and dissolve 10 mgO ~K of activity of 150 I.U~
per miligram.
1:1 . ,
- 12 -'~PIPrS = plpERAzrN~ [BIS] ETHANE SULFONIC ACID
14 I .
15 I IX
16 ¦ Sta~ilization of creatine, ATP and ~EP
17 ¦ 1, non ml of water
18 ¦ Add 12.1 ~rams of tris(hydroxymethyl) aminomethane
l~ ¦ Add 1.0 grams gelatin
20 ¦ Dissolve with heat above 30~C
21 ¦ Cool to room temperature
22 ¦ Add 2.0 grams ATP :
23 ¦ Add 2 grams P~P
24 ¦ . Add 10.0 grams creatine
2~ Dissolve and adjust pH to 9
271
2~1
29
31 I
32 ~ 3-
~ ll(lZZZ~i ~
2 To the stabilized solution of Example IX,
3 100 I.U./liter to 10,000 I.U./liter of LDH was added
4 and
100 I.U./liter to 10,000 I.U./liter of PK was added,
6 prior to packaging.
8 Each of the compositions of Examples V through X have
9 the same long shelf life without any substantial degradation.
0 Moreover, each of the above examples are based on samples
11 actually prepared and tested in accordance with the present
12 invention.
~3
1~
1~
16
17
18
19
21 I . I
~2
23
27
30~ ~
32 li
-24-
!i
