CA1102225A - Stabilized liquid enzyme and coenzyme compositions and method of preparing same - Google Patents
Stabilized liquid enzyme and coenzyme compositions and method of preparing sameInfo
- Publication number
- CA1102225A CA1102225A CA285,845A CA285845A CA1102225A CA 1102225 A CA1102225 A CA 1102225A CA 285845 A CA285845 A CA 285845A CA 1102225 A CA1102225 A CA 1102225A
- Authority
- CA
- Canada
- Prior art keywords
- coenzyme
- composition
- further characterized
- stabilized
- enzyme
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
- C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
- C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
- C07D307/56—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D307/62—Three oxygen atoms, e.g. ascorbic acid
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J9/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/008—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions for determining co-enzymes or co-factors, e.g. NAD, ATP
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
- C12Q1/50—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving creatine phosphokinase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/54—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving glucose or galactose
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- Immunology (AREA)
- General Engineering & Computer Science (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biophysics (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Medicinal Chemistry (AREA)
- Urology & Nephrology (AREA)
- Cell Biology (AREA)
- Emergency Medicine (AREA)
- Food Science & Technology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
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.
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.
Description
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.
1, i zz5 2 It is, therefore, the primary object of the present inven-3 tion to provide a liquid composition with coenzymes and/or
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.
1, i zz5 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 1, ', !l ~ 22S
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.
J
!
~ llU~ZZ5 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.
i ;
1 ~2~:5 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:
li LDH .
12 LACTATE + NAD ~ ~ NADH2 + PYRUVATE
14 Conversely, more than the three reactions listed may be involved, as in the case of Creatine phosphokinase (CPK):
CPK
18 (1) GP + ADP C ~ ATP + CREATINE
HK
21 (2) ATP + GLUCOSE < > GLUCOSE-6-PHOS. + ADP
2~ (3) GLUCOSE-6-PHOS. + NAD ~_ _ ~ NADH2 PMS
27 (4) NADH2 + INT ~ ~ INT + NAD
28 (ox) (.red) 33l ~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 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 ~
.~ -8-~ .
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.
GLUCOSE + ~TP ~ G-6~P + ADP
l G-6-P~H
61 G-6-P + NAD < ~ NADH ~ 6-PHOSPHOGLUCONIC ACID
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 I -10- .
11(~2ZZ5 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 ~, -11-., 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 1l -12-I ., !
~ 2~i 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 llO~;~Z~
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.
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.
~lCiZZZ5 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 ~`
` 31 ~2 ~16-!¦ ' ~ ~1(12Z25 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
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 1, ', !l ~ 22S
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.
J
!
~ llU~ZZ5 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.
i ;
1 ~2~:5 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:
li LDH .
12 LACTATE + NAD ~ ~ NADH2 + PYRUVATE
14 Conversely, more than the three reactions listed may be involved, as in the case of Creatine phosphokinase (CPK):
CPK
18 (1) GP + ADP C ~ ATP + CREATINE
HK
21 (2) ATP + GLUCOSE < > GLUCOSE-6-PHOS. + ADP
2~ (3) GLUCOSE-6-PHOS. + NAD ~_ _ ~ NADH2 PMS
27 (4) NADH2 + INT ~ ~ INT + NAD
28 (ox) (.red) 33l ~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 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 ~
.~ -8-~ .
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.
GLUCOSE + ~TP ~ G-6~P + ADP
l G-6-P~H
61 G-6-P + NAD < ~ NADH ~ 6-PHOSPHOGLUCONIC ACID
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 I -10- .
11(~2ZZ5 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 ~, -11-., 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 1l -12-I ., !
~ 2~i 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 llO~;~Z~
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.
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.
~lCiZZZ5 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 ~`
` 31 ~2 ~16-!¦ ' ~ ~1(12Z25 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 22 The remaining reactions are all self explanatory with reference 23 to the list of symbols set forth above, For an NA~P reaction, G-6-P + NADP ~ NADPH + 6~phosphogluconic acid ~ 2 ~ Z 5 l For an ADP reaction, 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, lO ADP + PEP ~ ATP + Pyruvate ~1 12 Pyruvate + NADH ~ > Lactate + ~IAD
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.
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--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.
18 ` CK
19 CREATINE + ATP ~ CP + ADP
C
pH 8-9 22 The remaining reactions are all self explanatory with reference 23 to the list of symbols set forth above, For an NA~P reaction, G-6-P + NADP ~ NADPH + 6~phosphogluconic acid ~ 2 ~ Z 5 l For an ADP reaction, 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, lO ADP + PEP ~ ATP + Pyruvate ~1 12 Pyruvate + NADH ~ > Lactate + ~IAD
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.
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--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.
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.
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.
--:ZO--()Z;Z~5 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
~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.
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.
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.
--:ZO--()Z;Z~5 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.
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 31 ~
, , 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~
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.
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 ~z~
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 .
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 2~1 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
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.
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 31 ~
, , 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~
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.
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 ~z~
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 .
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 2~1 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~
21 I . I
~2 30~ ~
32 li !i
~3 1~
1~
21 I . I
~2 30~ ~
32 li !i
Claims (32)
1. A stabilized liquid enzyme and coenzyme composition used in biological diagnostic determinations and which enzyme and coenzyme are normally unstable in an aqueous media, said composition comprising:
a) an aqueous vehicle, b) at least a sufficient amount of coenzyme to perform a determination dissolved in said aqueous vehicle, c) at least 100 I.U. of enzyme dissolved in said aqueous vehicle and both said enzyme and coenzyme cooperating in a determination reaction, d) at least 5% V/V of a non-reactive aqueous miscible organic solvent in said aqueous vehicle and which is liquid at least at room temperature, e) and said composition having a pH from about 6.0 to about 8.5, such that the enzyme and coenzyme are stabilized, f) said enzyme being selected from the class consisting of glucose-6-phosphate dehydrogenase, hexokinase, glutamate dehydrogenase, creatine phosphokinase, pyruvate kinase and alkali phosphatase, and said coenzyme being selected from the class con-sisting of nicotinamide-adenine dinucleotide, adenosin triphosphate, adenosin-5'-diphosphate, nicotinamide-adenine dinucleotide phosphate, and adenosin monophosphate.
a) an aqueous vehicle, b) at least a sufficient amount of coenzyme to perform a determination dissolved in said aqueous vehicle, c) at least 100 I.U. of enzyme dissolved in said aqueous vehicle and both said enzyme and coenzyme cooperating in a determination reaction, d) at least 5% V/V of a non-reactive aqueous miscible organic solvent in said aqueous vehicle and which is liquid at least at room temperature, e) and said composition having a pH from about 6.0 to about 8.5, such that the enzyme and coenzyme are stabilized, f) said enzyme being selected from the class consisting of glucose-6-phosphate dehydrogenase, hexokinase, glutamate dehydrogenase, creatine phosphokinase, pyruvate kinase and alkali phosphatase, and said coenzyme being selected from the class con-sisting of nicotinamide-adenine dinucleotide, adenosin triphosphate, adenosin-5'-diphosphate, nicotinamide-adenine dinucleotide phosphate, and adenosin monophosphate.
2. The stabilized liquid composition of claim 1 further characterized in that said composition comprises a first labile coenzyme and at least one second labile coenzyme which is also stabilized by at least said organic solvent.
3. The stabilized liquid composition of claim 1 further characterized in that said composition comprises a water soluble polymer which does not substantially inhibit enzymatic activity.
4. The stabilized liquid composition of claim 3 further characterized in that said composition comprises a first labile enzyme and at least one second labile enzyme which is also stabilize by at least said solvent and said polymer.
5. The stabilized liquid composition of claim 1 further characterized in that said composition comprises a bacteriostat which provides stabilization as well as providing bacteriostatic action.
6. The stabilized liquid composition of claim 5 further characterized in that the bacteriostat is an azide compound.
7. The stabilized liquid composition of claim 1 further characterized in that said solvent has the following character-istics:
a) pH of 4 to 10;
b) Liquid at room and refrigerator temperatures;
c) Does not react with the coenzymes or 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).
a) pH of 4 to 10;
b) Liquid at room and refrigerator temperatures;
c) Does not react with the coenzymes or 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).
8. The stabilized liquid composition of claim 1 further characterized in that said composition comprises at least two coenzymes and at least two enzymes.
9. A stabilized liquid coenzyme composition used in biological determinations which coenzyme is normally unstable in an aqueous media, said composition comprising a) at least 30% V/V of a non-reactive aqueous vehicle, b) at least a sufficient amount of coenzyme to perform a determination dissolved in said aqueous vehicle and cooperat-ing in a determination reaction, c) an aqueous miscible organic solvent in said aqueous vehicle and which is liquid at least at room temperature, and d) said composition having a pH of from about 6.0 to about 8.5, such that the coenzyme is stabilized, e) said coenzyme being selected from the class consisting of nicotinamide-adenine dinucleotide, adenosin triphosphate, adenosin-5'-diphosphate, creatine phosphokinase, nicotinamide-adenine dinucleotide phosphate, and adenosin monophosphate.
10. The stabilized liquid coenzyme composition of claim 9 further characterized in that said composition comprises a water soluble polymer which does not substantially inhibit enzymatic activity and that said composition also comprises a labile second coenzyme which is also stabilized by at least said organic solvent or said polymer.
11. The stabilized liquid coenzyme composition of claim 9 further characterized in that said composition comprises a bacteriostat which provides stabilization as well as providing bacteriostatic action.
12. The stabilized liquid coenzyme composition of claim 9 further characterized in that the organic solvent is non-reactive with said coenzyme and aqueous vehicle at room and refrigerator temperatures.
13. The stabilized liquid coenzyme composition of claim 9 further characterized in that the coenzyme is selected from the class consisting of NAD, ATP, ADP, CK, CP and NADP.
14. The stabilized liquid coenzyme composition of claim 9 further characterized in that the coenzyme is NAD with a concentration of above 1.2 grams per liter of liquid composition.
15. The stabilized liquid coenzyme composition of claim 9 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 coenzymes other than forming electrostatic (i.e., hydrogen) bonds;
d) Miscible with water;
e) Standard free energy of solvolysis is low (normal resonance is established).
a) pH between 4 to 10;
b) Liquid at room and refrigerator temperatures;
c) Does not react with coenzymes other than forming electrostatic (i.e., hydrogen) bonds;
d) Miscible with water;
e) Standard free energy of solvolysis is low (normal resonance is established).
16. The stabilized liquid coenzyme composition of claim 15 further characterized in that the organic solvent is a polyol.
17. A method of stabilizing a labile coenzyme and labile enzyme used in biological diagnostic determinations and which enzyme and coenzyme are normally unstable in aqueous media, said method comprising a) mixing water with an aqueous miscible non-reactive organic solvent to form an aqueous miscible organic solvent solution and which organic solvent is liquid at least at room temperature, b) dissolving a polymer in the aqueous miscible organic solvent solution, c) adding at least a sufficient amount per liter of a labile coenzyme to said solution to perform a determination and which is dissolved in said solution and cooperates in a determination reaction, d) adjusting the pH to within the range of 6.0 to 8.5, such that the coenzyme is stabilized, e) adding at least 100 I.U. per liter of a labile enzyme to said solution, and which enzyme is dissolved in said solution and cooperates in a determination reaction, and f) sealing the composition, g) said enzyme being selected from the class consisting of glucose-6-phosphate dehydrogenase, hexokinase, glutamate dehydrogenase, creatine phosphokinase, pyruvate kinase and alkali phosphatase, and said coenzyme being selected from the class consisting of nicotinamide-adenine dinucleotide, adenosin triphosphate, adenosin-5'-diphosphate, nicotinamide-adenine dinucleotide phosphate, and adenosin monophosphate.
18. The method of claim 17 further characterized in that said method comprises adding a bacteriostatic agent which also functions as an enzyme stabilizing agent.
19. The method of claim 18 further characterized in that said bacteriostatic agent is an azide compound.
20. The method of claim 17 further characterized in that said method also comprises adding a second coenzyme to said solution which is also stabilized therein.
21. The method of claim 17 further characterized in that said method also comprises adding a second enzyme to said solution which is also stabilized therein, after adjustment of the pH.
22. The method of claim 17 further characterized in that said solvent has the following characteristics:
a) pH between 4 to 10;
b) liquid at room and refrigerator temperatures;
c) does not react with the enzymes or coenzymes other than forming electrostatic (i.e., hydrogen) bonds;
d) miscible with water;
e) standard free energy of solvolysis is low (normal resonance is established).
a) pH between 4 to 10;
b) liquid at room and refrigerator temperatures;
c) does not react with the enzymes or coenzymes other than forming electrostatic (i.e., hydrogen) bonds;
d) miscible with water;
e) standard free energy of solvolysis is low (normal resonance is established).
23. The method of claim 22 further characterized in that the organic solvent is a polyol which contains 2-4 hydroxyl groups and 2-10 carbon atoms.
24. The method of claim 17 further characterized in that said solvent is added so that it is present in an amount of about 25% to about 40% by volume.
25. The method of claim 17 further characterized in that said polymer is present in an amount of at least 0.01%.
26. A method of stabilizing a labile coenzyme used in biological diagnostic determinations and which coenzyme is normally unstable in an aqueous media, said method comprising a) dissolving a coenzyme in an aqueous base in an amount sufficient to perform a determination and which coenzyme cooperates in a determination reaction, b) contacting said coenzyme containing aqueous base with at least 5% by volume of a non-reactive aqueous miscible organic solvent to provide a stabilized composition and which solvent is liquid at least at room temperature, c) adjusting the composition pH to about 6.0 to about 8.5, such that the coenzyme is stabilized, d) said coenzyme being selected from the class consist-ing of nicotinamide-adenine dinucleotide, adenosin triphosphate, adenosin-5'-diphosphate, creatine phosphokinase, nicotinamide-adenine dinucleotide phosphate, and adenosin monophosphate, e) sealing the composition in a container.
27. The method of claim 26 further characterized in that said method comprises dissolving a water soluble polymer in said composition which does not substantially inhibit enzymatic activity.
28. The method of claim 26 further characterized in that said method also comprises adding a second coenzyme to said solution which is also stabilized therein.
29. The method of claim 26 further characterized in that said solvent has the following characteristics:
a) pH between 4 to 10;
b) liquid at room and refrigerator temperatures;
c) does not react with coenzymes other than forming electrostatic (i.e., hydrogen) bonds;
d) miscible with water;
e) standard free energy of solvolysis is low (normal resonance is established).
a) pH between 4 to 10;
b) liquid at room and refrigerator temperatures;
c) does not react with coenzymes other than forming electrostatic (i.e., hydrogen) bonds;
d) miscible with water;
e) standard free energy of solvolysis is low (normal resonance is established).
30. The method of claim 26 further characterized in that said solvent is added so that it is present in an amount of about 25% to about 50% by volume.
31. The method of claim 29 further characterized in that the solvent is a liquid polyol containing from 2-10 carbon atoms and 2-4 hydroxyl groups.
32. The method of claim 27 further characterized in that the polymer is geltain present in said solution in an amount of at least 0.01%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA372,912A CA1127055A (en) | 1976-09-13 | 1981-03-12 | Stabilized liquid enzyme and coenzyme compositions and method of preparing same |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US72256576A | 1976-09-13 | 1976-09-13 | |
| US722,565 | 1976-09-13 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1102225A true CA1102225A (en) | 1981-06-02 |
Family
ID=24902389
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA285,845A Expired CA1102225A (en) | 1976-09-13 | 1977-08-31 | Stabilized liquid enzyme and coenzyme compositions and method of preparing same |
Country Status (7)
| Country | Link |
|---|---|
| JP (2) | JPS5843078B2 (en) |
| CA (1) | CA1102225A (en) |
| CH (1) | CH631208A5 (en) |
| DE (1) | DE2740957A1 (en) |
| FR (1) | FR2364457A1 (en) |
| GB (1) | GB1570971A (en) |
| SE (1) | SE446742B (en) |
Families Citing this family (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA1102225A (en) * | 1976-09-13 | 1981-06-02 | Ivan E. Modrovich | Stabilized liquid enzyme and coenzyme compositions and method of preparing same |
| DE2814154A1 (en) * | 1978-04-01 | 1979-10-11 | Behringwerke Ag | STABILIZED NICOTINAMIDE NUCLEOTIDES AND METHOD FOR MANUFACTURING THEM |
| JPS5513008A (en) * | 1978-07-11 | 1980-01-29 | Mitsui Toatsu Chem Inc | Stabilization of aqueous solution of enzyme |
| CA1187388A (en) * | 1978-09-20 | 1985-05-21 | American Monitor Corporation | Stabilization of working reagent solutions containing nadh, nadph, and/or enzymes, and the use of such stabilized reagents in enzymes or substrate assays |
| JPS55138399A (en) * | 1979-04-13 | 1980-10-29 | Dainippon Pharmaceut Co Ltd | Composition for determining beta-d-galactosidase and method thereof |
| JPS5982398A (en) * | 1982-11-01 | 1984-05-12 | Toyobo Co Ltd | Method for stabilizing coenzyme |
| JPS6095746U (en) * | 1983-12-08 | 1985-06-29 | 株式会社ニツコー | Radio control toy radio transmitter |
| JPS62101773A (en) * | 1985-10-29 | 1987-05-12 | 白木金属工業株式会社 | Electronic key apparatus |
| US5298406A (en) * | 1992-09-14 | 1994-03-29 | E. I. Du Pont De Nemours And Company | Formulation for stabilizing enzymatic activity and immunoreactivity of creatine kinase and creatine kinase isoenzymes |
| DE10313972A1 (en) * | 2003-03-27 | 2004-10-21 | Degussa Ag | Coupled cofactor-dependent enzymatic reaction system |
| US7879567B2 (en) * | 2004-10-05 | 2011-02-01 | Asahi Kasei Pharma Corporation | Method for stabilizing coenzyme and composition therefor |
Family Cites Families (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1084079A (en) * | 1964-11-30 | Beckman Instruments Inc | ||
| DE1673194A1 (en) * | 1965-07-22 | 1970-10-29 | Dresden Arzneimittel | Method and means for the determination of transaminase activities |
| US3296094A (en) * | 1966-05-05 | 1967-01-03 | Baxter Laboratories Inc | Stabilized aqueous enzyme solutions |
| US3557002A (en) * | 1967-11-15 | 1971-01-19 | Procter & Gamble | Stabilized aqueous enzyme preparation |
| US3627688A (en) * | 1968-11-12 | 1971-12-14 | Procter & Gamble | Stabilized aqueous enzyme containing compositions |
| GB1322951A (en) * | 1969-10-29 | 1973-07-11 | Warner Lambert Co | Process and agent for the determination of glucose |
| DE2061984C3 (en) * | 1970-12-16 | 1974-04-11 | Boehringer Mannheim Gmbh, 6800 Mannheim | Reagent for the determination of lactate dehydrogenase in the color test |
| DE2302721C2 (en) * | 1973-01-19 | 1975-01-23 | Boehringer Mannheim Gmbh, 6800 Mannheim | Method for the determination of creatine kinase |
| US3920798A (en) * | 1973-05-08 | 1975-11-18 | Union Carbide Corp | Zeolite y synthesis |
| US3962037A (en) * | 1975-01-30 | 1976-06-08 | Miles Laboratories, Inc. | Composition for stabilizing an enzyme-containing reagent |
| NL7603588A (en) * | 1975-04-21 | 1976-10-25 | Hoffmann La Roche | STABILIZED COENZYME SOLUTION. |
| JPS52111183A (en) * | 1976-03-12 | 1977-09-17 | Hitachi Ltd | Hand rail guiding device |
| FR2344570A1 (en) * | 1976-03-17 | 1977-10-14 | Modrovich Ivan | PROCESS FOR STABILIZING A LABILE COENZYME |
| CA1091174A (en) * | 1976-03-17 | 1980-12-09 | Ivan E. Modrovich | Stabilized liquid enzyme |
| CA1102225A (en) * | 1976-09-13 | 1981-06-02 | Ivan E. Modrovich | Stabilized liquid enzyme and coenzyme compositions and method of preparing same |
-
1977
- 1977-08-31 CA CA285,845A patent/CA1102225A/en not_active Expired
- 1977-09-08 GB GB3744577A patent/GB1570971A/en not_active Expired
- 1977-09-09 FR FR7727386A patent/FR2364457A1/en active Granted
- 1977-09-12 DE DE19772740957 patent/DE2740957A1/en active Granted
- 1977-09-12 CH CH1111877A patent/CH631208A5/en not_active IP Right Cessation
- 1977-09-13 JP JP52111183A patent/JPS5843078B2/en not_active Expired
- 1977-10-20 SE SE7711853A patent/SE446742B/en not_active IP Right Cessation
-
1984
- 1984-09-29 JP JP20545084A patent/JPS60105494A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| FR2364457B1 (en) | 1980-04-25 |
| FR2364457A1 (en) | 1978-04-07 |
| JPH0147999B2 (en) | 1989-10-17 |
| JPS5843078B2 (en) | 1983-09-24 |
| JPS5352685A (en) | 1978-05-13 |
| CH631208A5 (en) | 1982-07-30 |
| SE446742B (en) | 1986-10-06 |
| DE2740957A1 (en) | 1978-03-16 |
| SE7711853L (en) | 1979-04-21 |
| GB1570971A (en) | 1980-07-09 |
| JPS60105494A (en) | 1985-06-10 |
| DE2740957C2 (en) | 1987-07-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US4250254A (en) | Stabilized liquid enzyme and coenzyme compositions | |
| US4271264A (en) | Stabilized liquid enzyme and coenzyme compositions | |
| CA1178875A (en) | Stabilization of coenzymes in aqueous solution | |
| US4277562A (en) | Stabilized liquid enzyme and coenzyme compositions | |
| CA1102225A (en) | Stabilized liquid enzyme and coenzyme compositions and method of preparing same | |
| US4372874A (en) | Stabilization of hydrolysis prone labile organic reagents in liquid media | |
| US3860484A (en) | Enzyme stabilization | |
| US3764478A (en) | Stabilized enzymatic test reagents | |
| US4652524A (en) | Soluble stabilized enzymes | |
| EP0072581A1 (en) | The stabilization of working reagent solutions containing enzymes, and the use of such stabilized reagents in enzyme or substrate assays | |
| US4310625A (en) | Stabilized liquid enzyme compositions for diagnostic determinations | |
| CA1092035A (en) | Liquid stabilized coenzyme | |
| US4189536A (en) | Reagent systems, enzymatic assays and compositions therefor | |
| CA1091174A (en) | Stabilized liquid enzyme | |
| US3974037A (en) | Process for measuring carbon dioxide content of the body fluid | |
| US4339533A (en) | Stabilization of creatine kinase (CK) and its application as a reference standard | |
| US4132598A (en) | Stabilized liquid phosphate containing diagnostic compositions and method of preparing same | |
| CA1127055A (en) | Stabilized liquid enzyme and coenzyme compositions and method of preparing same | |
| US5266472A (en) | Stabilization of the enzyme urate oxidase in liquid form | |
| US4229369A (en) | Tris (hydroxymethyl) aminomethane salt of 2-mercaptosuccinic acid | |
| US4310624A (en) | Stabilized liquid coenzyme compositions for diagnostic determinations | |
| US4219645A (en) | Stabilized nicotinamide-adenine dinucleotides and process for their preparation | |
| CA1132034A (en) | Stabilization of hydrolysis prone labile organic reagents in liquid media | |
| US5188955A (en) | Method of preserving arylacylamidase in aqueous solution | |
| CA2183573A1 (en) | Isoenzyme calibrator/control products |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| MKEX | Expiry |