WO2012050536A1

WO2012050536A1 - Method of the adenosine diphosphate quantitative determination

Info

Publication number: WO2012050536A1
Application number: PCT/SK2010/050018
Authority: WO
Inventors: Miroslav Stredansky; Silvia Stredanska; Pavol Szomolanyi
Original assignee: Biorealis, S.R.O.
Priority date: 2010-10-15
Filing date: 2010-10-15
Publication date: 2012-04-19

Abstract

The present invention provides an innovative method for the determination of ADP. The method is based on the phosphorylation of ADP with creatine kinase and creatine phosphate forming creatine which is further transformed with creatinase to sarcosine followed by sarcosine oxidation with sarcosine oxidase in the presence of an electron- acceptor. The method is suitable for the determination of ADP in the presence of ATP, for the determination of AMP after its transformation to ADP by myokinase, for the determination of cyclic AMP after its transformation to ADP by cyclic-3',5'-nucleotide phosphodiesterase and myokinase, for the determination of activities, substrates and inhibitors of the kinases and other involved enzymes. The enzymes used in this invention are stable and available and can be applied in free or immobilised form. The signal proportional to the amount of ADP is detected by electrochemical or optical methods.

Description

Method of the adenosine diphosphate quantitative determination

Field of the invention

This invention relates to chemical and biochemical analyses, particularly, this invention concerns a way of a quantitative detection of adenosine diphosphate (hereinafter abbreviated as ADP) using a coupled enzymatic reactions in various formats. This invention is especially advantageous for the determination of ADP in the presence of adenosine triphosphate (hereinafter abbreviated as ATP), for the determination of activities of the enzymes producing and consuming ADP, such as kinases, for the determination of the reaction substrates and inhibitors of the kinases and other involved enzymes.

Background of the invention

Adenine nucleotides exhibit a great importance in the energy metabolism of living organisms. There is a long interest in the quantitative detection of ADP for diagnosis of various diseases and in the quantitative detection of the reactions yielding ADP, especially from ATP. The accurate measurement of ADP allows the quantitative determination of enzymatic activities of enzymes producing or consuming ADP by their reaction and products or substrates of these enzymes.

Methods based on various principles have been developed for the ADP measurements. High performance liquid chromatography (HPLC) has been employed for the determination of adenosine nucleotides (Anderson and Murphy, J. Chromatogr. 121, pp. 251-262, 1976; Liu et al, Food Technol. Biotechnol. 44, pp.531-534, 2006). Nuclear magnetic resonance (NMR) spectroscopy has been used for the direct determination of ADP in the presence of ATP ( Petersen et al., Biochim. Biophys. Acta 1035, pp. 169-174, 1990; Ben-Bashat et al., J. Magn. Reson. 110, pp. 231-239, 1996). However these methods are time-consuming and require expensive instruments and specialised personnel.

Enzymatic methods for the ADP detection are often more simple. A bioluminiscent method is a well-established technique for this purpose. Several variants of this technique for the quantitative determination of ADP and other nucleotides have been described (e.g. US Patent 5891659; Girotti et al., Anal. Biochem. 192, pp. 350-357, 1991; Gorman et al., Luminiscence 18, pp.173-181, 2003). In principle, ADP is converted to ATP by pyruvate kinase in the presence of phosphoenolpyruvate (hereinafter abbreviated as PEP). ATP then react with luciferine and oxygen in the presence of luciferinase to emit light measured by a luminometer. Although this method is widely used, its suitability for the measurements of the kinase activity and of the products or substrates of these enzymes is very limited, because the added ATP is interfering measurements.

Marquette et al. (Anal. Bioanal. Chem. 377, pp. 922-928, 2003) has presented a luminescent biochip for acetate monitoring based on the ADP measurement. In this case acetate reacted with ATP using acetate kinase forming ADP. ADP reacted with PEP using pyruvate kinase to form pyruvate which was oxidised with pyruvate oxidase to form hydrogen peroxide. The amount of hydrogen peroxide was detected by electrochemiluminiscent measurement in the presence of immobilised luminol. ATP does not interfere the measurement. However the system is complicated and the measurements suffer from high errors.

US Patent 5916761 describes a method for determining ADP using hexokinase, oxidized nicotinamide adenine dinucleotide (phosphate) [hereinafter abbreviated as NAD(P)], and glucose-6-phosphate dehydrogenase. The amount of the reduced NAD(P) proportional to the amount of ADP is measured by UV spectrophotometry. An disadvantage of this method is a necessity of the unstable NAD(P) cofactor for the assay.

Another way for the quantitative determination of ADP is described in US Patent 4923796. In principle ADP is phosphorylated with kinase enzyme (e.g. galactokinase, formate kinase, pyruvate kinase) in the presence of the corresponding phosphorylated substrate (e.g. galactose- 1 -phosphate, formyl-phosphate, PEP). The dephosphorylated substrate (e.g. galactose, formate, pyruvate) is reacted with the corresponding dehydrogenase (e.g. galactose dehydrogenase, formate dehydrogenase, pyruvate dehydrogenase) to form reduced form of cofactor NADH from NAD. Subsequently NADH . is measured by UV spectrophotometry or it is reacted with tetrazolium salt in the presence of diaphorase to form coloured formazan measured by spectrophotometry. An disadvantage is a necessary presence of unstable phosphorylated substrates and the NAD cofactor.

US Patent 7410755 describes the method for determining ATPase activity and determining ADP in the presence of ATP by fluorescent or chemiluminiscent measurements. In principle ADP reacts with PEP using pyruvate kinase to form pyruvate which is oxidised with pyruvate oxidase to form hydrogen peroxide. Subsequently hydrogen peroxide in the presence of peroxidase oxidises specific dyes to create fluorescent or chemiluminiscent signal proportional to the amount of ADP. As this system suffers from adventitious contamination of the assay medium introducing erroneous results, an addition of correcting components during the analysis (such as creatine phosphokinase and phosphocreatine, pyruvate kinase and PEP, peroxidase and non-interfering peroxidase substrate, and catalase) is necessary. So the analysis becomes really complicated.

The measurement of ADP has been used also for the determination of the reaction substrates of the kinases. A typical example is acetate. One design of the acetate determination is based on the reaction of acetate with ATP using acetate kinase forming ADP followed by reaction of ADP with PEP using pyruvate kinase to form pyruvate, which oxidises NADH to NAD using lactate dehydrogenase. The depletion of NADH is monitored either by UV spectroscopy (Trivin et al., Clin. Chim. Acta 121, pp. 43-50, 1982) or electrochemically (Tang et al., Biotechnol. Techniques 1 1, pp. 683-687, 1997). The necessity of very unstable NADH introduces practical problems.

Another method determination of acetate is based on the oxidation of pyruvate (formed from acetate as described above) with molecular oxygen using pyruvate oxidase. The depletion of oxygen is monitored by oxygen electrode electrochemically (Mizutani et al., Sensors Actuators B 91, pp. 195-198, 2003). A drawback of this method is a high instability of pyruvate oxidase and its activators, thiamine pyrophosphate (TPP) and flavin adenine dinucleotide (FAD), which are also very expensive.

On the other hand, enzymatic methods for the determination of creatinine and creatine are known for many years (Fossati et al., Clin. Chem. 29, pp. 1494-1496, 1983; Lindback and Bergman, Clin. Chem. 35, pp. 835-837, 1989; Killard and Smyth, Trends Biotechnol. 18, 433-437, 2000). In principle creatinine is hydrolysed by creatininase to creatine which is further hydrolysed by creatinase to sarcosine. Sarcosine is subsequently oxidised by sarcosine oxidase and this reaction is monitored by various physicochemical ways.

Brief description of the drawings

Figure 1 : calibration curve for ADP determined by the chemiluminiscent method using a luminometer.

Figure 2 : calibration curve for ADP determined amperometrically by the Clark oxygen electrode. Figure 3 : calibration curve for acetate determined spectrophotometrically by the absorbance measurement.

Figure 4 : calibration curve for oleate determined by fluorescent method using a fluorometer.

Figure 5 : calibration curve for hexoses determined amperometrically using the multienzyme biosensor.

Figure 6 : calibration curve for caffeine determined amperometrically using the multienzyme biosensor.

Detailed description of the invention

The method of the ADP determination described by this invention overcomes many of the problems outlined above. The method requires stepwise or simultaneous action of several enzymes and at least one additive reaction substrate: creatine phosphate (CP). The sequence of enzymatic reactions included in this method is following:

a) a phosphorylation of ADP with creatine kinase in the presence of creatine phosphate forming ATP and creatine;

b) a hydrolysis of creatine with creatinase forming urea and sarcosine; c) an oxidation of sarcosine with sarcosine oxidase in the presence of an electron-acceptor.

These enzymatic reactions are coupled with a physicochemical detection. Several parameters proportional to the concentration (amount) of ADP can be detected: the depletion of oxygen which is a natural electron-acceptor, the depletion of another electron- acceptor, the increase of hydrogen peroxide when oxygen is the electron-acceptor, and the increase of the reduced form of the other electron-acceptor. Variety ways of performing measurement can be applied. Preferably said measurement is selected from endpoint assay, kinetic mode assay, flow-injection analysis.

The present invention includes also the reagents for the determination of ADP, which reagents comprise creatine kinase, creatine phosphate, creatinase, sarcosine oxidase, and optionally other substances improving the measurement, such as enzyme activators, enzyme stabilisers, surfactants, electron-acceptors, and other enzymes. The reagent according the present invention can be in dry form, in the form of solution, suspension, paste, film, layer, membrane. The reagent can be also impregnated on to an appropriate carrier or can be incorporated into a test-strip, on a surface electrode or optode.

The method of the present invention is particularly suitable for the determining ADP in liquid samples. The enzymes involved in this method are stable and available and can be applied in free form or immobilised at suitable conditions.

When said enzymes are used in the free soluble form, the reaction mixture can contain all enzymes together with CP, or the enzymes can be added stepwise in the order: creatine kinase, creatinase, sarcosine oxidase. Preferably all enzymes with CP are applied together with a measured sample containing ADP and a change of a physicochemical property of the mixture is measured at suitable conditions.

Said suitable conditions mean above all the suitable temperature and pH. The temperature is important factor. It should be kept in the range acceptable for all present enzymes. The suitable temperature range is from 5 °C to 50 °C, preferably from 15 °C to 40 °C. Said suitable pH value of the reaction mixture of the present invention can be kept constant in the range acceptable for all present enzymes using buffering salts. The suitable pH range is from 5 to 9.5, preferably from 6.5 to 8. Various buffering salts can be used for the method of the present invention. The preferred buffers include phosphate, Hepes, Tris, citrate, imidazol, MOPS, borate, succinate, acetate, pyrophosphate.

Said enzyme activators are selected from the compounds containing Mg²⁺, Ca²⁺, K⁺, Mn²⁺, Co²⁺, Cu⁺, Zn²⁺, acetate, tartrate, succinate, citrate, glyoxalate, dithio-threitol.

Said enzyme stabilisers are selected from the group of sugars, polyols, aminoacids and organic salts, such as sucrose, lactose, trehalose, sorbitol, lactitol, glycerol, diethylene glycol, low molecular weight polyethylene glycols, glutamate, lysine, glycine, succinate, maleate.

Said surfactants are any natural or synthetic compound having detersive properties,, preferably selected from the group of biologically compatible neutral surfactants (e.g. Triton® X-100, Tween®, Tergitol®, Brij®, Span®, polyethylene glycol, polyethylene oxide, polypropylene oxide), anionic detergents (e.g. sodium dodecylsulphate, cholate, taurocholate, phospholipide) and cationic surfactants (e.g. cetyltrimethylammonium bromide).

The selection of said electron-acceptor depends on the way of the physicochemical detection. According one embodiment of the present invention, when the detection is performed by optical methods, said electron-acceptor is the compound which change optical properties (increase or decrease of light absorbance or emission) when is reduced by sarcosine oxidase. The preferred electron-acceptors are selected from the group of colorants, fluorescent dyes, chemiluminiscent substrates. More preferably, said electron- acceptor is selected from the group of methylene blue, thionine, neutral red, toluidine blue, Meldola's blue, ferricyanide, ferricinium and its derivates, resorufin derivates, rhodamines, coumarin derivates. When said electron-acceptor is oxygen, two ways of the optical detection are possible.

The first one is the employment of oxygen sensitive dyes, probes and optrodes, e.g. tris(2,2'-bipyridyl dichlororuthenium), platinum octaethylporphyrin and other metaloporphyrins. The second one is the detection of hydrogen peroxide formed by the reaction of sarcosine with oxygen. The detection of hydrogen peroxide by optical methods is well known in the art. In this case an additive enzyme peroxidase is involved together with an electron-donor which is oxidised with hydrogen peroxide to change optical properties. Said electron-donor is selected from the group of spectrophotometnc peroxidase substrates, fluorescent dyes, chemiluminiscent substrates. Preferably, said electron-donor is selected from the group of 4-amino-antipyrine, polyphenolic compounds (e.g. pyrogallol, catechol), phenylenediamine, diaminobenzidine, 2,2'-azino-bis(3-ethylbenzthiazoline- sulfonic acid), 4-aminophenazone, 3,5-dichloro-2-hydroxybenzenesulfonic acid, 3- hydroxy-2,4,6-triiodo benzoic acid, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine sodium salt, 2,4,6-tribromo-3-hydroxybenzoic acid, ferrocyanide, ferrocene and its derivates, resorufin derivates, rhodamines, coumarin derivates, fluorescein derivates, 2,4,5- triphenylimidazoles, 9-alkylidene-N-alkylacridans, 1 ,4-dioxenes, 1 ,4-thioxenes, 1,4- oxazines, arylimidazoles, 9-alkylidene-xanthenes and lucigenin.

According to another embodiment of the present invention, when the detection is performed by electrochemical methods, said electron-acceptor is the compound having, electroactive properties, it means giving measurable electrochemical signal when is reduced by sarcosine oxidase. Any electrochemical procedure known in art (e.g. potentiometric, amperometric, conductometric, coulometric) can be used for the measurement of said electrochemical signal, preferably the amperometric procedure is employed. The preferred electron-acceptors for said electrochemical detection are selected from the group consisting of cytochromes, quinones, aminophenols, electron acceptor aromatic compounds (e.g. tetrathiafulvalene and N-methylphenazinium), organic dyes (e.g. toluidine blue, Meldola's blue, neutral red, methylene blue and thionin), metallocenes, organometallic complexes of Os, Ru and V, inorganic complexes of Fe (e.g. ferricyanide).

When said electron-acceptor is oxygen, three methods of the electrochemical detection are possible. The first one is the employment of oxygen sensitive electrode (e.g. Clark electrode, gas permselective electrodes) for the measurement of the oxygen uptake which is proportional to the amount of ADP in the mixture. The second one is a direct electrochemical detection of hydrogen peroxide formed by the reaction of sarcosine with oxygen on the surface of the electrochemical working electrode. This electrode can be done from metal or carbon, preferably it has the active surface from platinum or from carbon nanotubes. The third one is the detection of hydrogen peroxide using an additive enzyme peroxidase together with an electron-donor which is oxidised with hydrogen peroxide to change measured electrochemical signal. Preferably, said electron-donor is selected from the group consisting of reduced cytochromes, phenolic compounds, aminophenols, electron-donor aromatic compounds (e.g. tetracyano-p-quinodimethane), organic dyes in reduced form, metallocenes (e.g. ferrocene and its denvates), organometallic complexes of Os, Ru and V, inorganic complexes of Fe (e.g. ferrocyanide).

Said electrochemical signals deriving from the reduction of said electron-acceptors or the oxidation of said electron-donors are measured by any electrochemical working electrode, preferably electrode having the active surface made from noble metals, carbon materials, conducting polymers.

According to the preferred embodiment of the present invention said enzymes and said other substances can be applied in the immobilised form using immobilisation procedures known in the art. Three measuring arrangements are preferred in this case. The first, when said enzymes are immobilised into the surface of inside of carrier particles (e.g. beads), the repeated batch analysis saving material costs can be performed. The second, the · flow injection analysis can be advantageously carried-out, when the material with said immobilised enzymes are packed in a column or bioreactor or said enzymes are immobilised into the inner surface of tubings or capillaries. The third, biosensors are prepared when said enzymes are immobilised in the vicinity of the surface or directly on the surface of physical tranducers, e.g. said electrochemical electrodes or optrodes. Said enzymes may be also incorporated in the electrode (optrode) material, where these electrodes (optrodes) are made from composite materials. All above mentioned substances, electrodes and optrodes are applicable also for biosensors. The biosensor arrangement is the most preferred embodiment of the present invention, because it allows rapid, accurate, specific, and convenient ADP determination.

According to the present invention, the method of the ADP determination is suitable also for the determination of AMP (adenosine monophosphate) after its transformation to ADP by myokinase. Myokinase (adenylate kinase) transforms one molecule of AMP and one molecule of ATP to two molecules of ADP which is determined as described above. So the determination of AMP according the present invention requires the presence of said myokinase, ATP, creatine kinase, creatine phosphate, creatinase, sarcosine oxidase and optionally said substances improving the measurement, such as enzyme activators, enzyme stabilisers, surfactants, electron-acceptors, other enzymes.

According to the present invention, the method of the ADP determination is suitable also for the determination of cyclic AMP (cAMP) after its transformation to ADP by cyclic-3', 5 '-nucleotide phosphodiesterase and myokinase. Cyclic-3 ',5 '-nucleotide phosphodiesterase hydrolyses a molecule of cAMP to AMP and subsequently myokinase transforms one molecule of AMP and one molecule of ATP to two molecules of ADP which is determined as described above. So the determination of AMP according the present invention requires the presence of said cyclic-3 ',5 '-nucleotide phosphodiesterase, myokinase, ATP, creatine kinase, creatine phosphate, creatinase, sarcosine oxidase and optionally said substances improving the measurement, such as enzyme activators, enzyme stabilisers, surfactants, electron-acceptors, other enzymes.

According to the present invention, the method of the ADP determination is suitable also for the determination of the enzymatic activity of kinases and the enzymes producing AMP by their catalytic action, e.g. cyclic-3 ',5 '-nucleotide phosphodiesterase and acyl-CoA synthetase. Kinases catalyse the phosphorylation of their specific substrates with a simultaneous transformation of ATP to ADP which is determined as described above. The measurement is preferably carried-out kinetically in the presence of ATP, kinase corresponding substrate, creatine kinase, creatine phosphate, creatinase, sarcosine oxidase and optionally said substances improving the measurement, such as enzyme activators, enzyme stabilisers, surfactants, electron-acceptors, other enzymes.

The kinase activity is commonly expressed in μπιοΐ of ADP forming per 1 min. In the following Table 1 are given examples of kinases which can be measured by the method of the present invention and their corresponding substrates. When the activity of the enzymes producing AMP by their catalytic action is determined, the measurement is preferably carried-out kinetically in the presence of ATP, corresponding substrate of the determining enzyme, myokinase, creatine kinase, creatine phosphate, creatinase, sarcosine oxidase and optionally other substances.

Table 1

According to the present invention, the method of the ADP determination is suitable also for the determination of substrates of kinases. Kinases catalyse the phosphorylation of their corresponding substrates with a simultaneous transformation of ATP to ADP which is determined as described above. So the determination of said substrates of kinases according the present invention requires the presence of said corresponding kinase, ATP, creatine kinase, creatine phosphate, creatinase, sarcosine oxidase and optionally said substances improving the measurement, such as enzyme activators, enzyme stabilisers, surfactants, electron-acceptors, other enzymes. In the Table 1 are given examples of said substrates of kinases which can be measured by the method of the present invention. In addition, the invention is suitable also for the determination of inhibitors of kinases.

According to the present invention, the method of the ADP determination is suitable also for the determination of substrates and inhibitors of the enzymes producing AMP by their catalytic action, e.g. cyclic-3', 5 '-nucleotide phosphodiesterase and acyl-CoA synthetase. So the determination of said substrates requires the presence of said corresponding enzyme producing AMP, myokinase, ATP, creatine kinase, creatine phosphate, creatinase, sarcosine oxidase and optionally said substances improving the measurement, such as enzyme activators, enzyme stabilisers, surfactants, electron- acceptors, other enzymes.

The subject matter of the present invention is illustrated by the following examples that do not limit the scope of protection.

Examples

Example 1

A concentration of ADP was determined by a chemiluminiscent measurement. The reaction mixture (1.8 ml) contained the reagents in 0.05 M phosphate buffer (pH 7.5) as follows: 2.5 U of creatine kinase (Sigma, St. Louis, USA, Cat. No. C 3755), 0.8 U of creatinase (Sigma, St. Louis, USA, Cat. No. C 2409), 1.5 U of sarcosine oxidase (Sigma, St. Louis, USA, Cat. No. S 7897 ), 5 U of peroxidase (Sigma, St. Louis, USA, Cat. No. P 8250), 2 mM creatine phosphate, 1 mM luminol, 1 mM p-iodophenol, 1.5 mM magnesium acetate. The ADP solutions (200 μΐ) of various concentrations were added to this reaction mixture. After 1 min the luminescent intensity was measured by luminometer at 430 nm. The calibration curve for ADP is illustrated in Fig. 1. Example 2

A concentration of ADP was determined by a measurement of oxygen depletion using an electrochemical oxygen electrode. The reaction mixture (2.9 ml) contained the reagents in 0.05 M phosphate buffer (pH 7.5) as follows: 4 U of creatine kinase, 1.2 U of creatinase, 2.5 U of sarcosine oxidase, 2 mM creatine phosphate, 1.5 mM magnesium chloride. The reaction mixture was placed into the 3-ml amperometric cell equipped with the Clark oxygen electrode (Amel, Milan, Italy). After the addition the ADP solution (100 μΐ) the cell was closed and the current change was measured. The calibration curve for ADP is illustrated in Fig. 2.

Example 3

A concentration of acetate (kinase substrate) was determined by a spectrophotometric measurement. The reaction mixture (1.9 ml) contained the reagents in 0.05 M phosphate buffer (pH 7.5) as follows: 2 U of acetate kinase (Sigma, St. Louis, USA, Cat. No. A 7437), 2.5 U of creatine kinase, 0.8 U of creatinase, 1.5 U of sarcosine oxidase, 5 U of peroxidase, 2 mM creatine phosphate, 2 mM 4-aminoantipyrine, 2 mM N-ethyl-N- (2-hydroxy-3-sulfopropyl)-m-toluidine sodium salt (Sigma, St. Louis, USA, Cat. No. E 8631), 1.5 mM magnesium chloride. The acetate solutions (100 μΐ) of various concentrations were added to this reaction mixture. Absorbance was measured by spectrophotometer at 550 nm. The calibration curve for acetate is illustrated in Fig. 3.

Example 4

Activities of glycerokinase preparations (Sigma, St. Louis, USA, Cat. No. G 4147 and G 6278) were determined by an amperometric biosensor. A platinum electrode (2 mm diameter; Amel, Milan, Italy)_, was cleaned and covered with an cellulose-acetate membrane. A solution of enzymes was prepared by mixing creatine kinase (8 mg/ml), creatinase (12 mg/ml), sarcosine oxidase ' (35 mg/ml ), peroxidase (5 mg/ml), and glutaraldehyde (0.5 mg/ml). 2 μΐ of this solution were placed into the surface of the membrane and left to dry for 3 h at room temperature followed by 15 min at 40 °C. The biosensor was immersed in 10 ml of 0.2M Tris buffer (pH 9.8) containing 5 mM ATP, 5 mM creatine phosphate, 2 mM magnesium acetate, 5 mM glycerol and a current was monitored at the constant potential of 700 mV against a reference electrode (standard calomel electrode, SCE). The biosensor was calibrated by successive additions of the ADP solution. The measurement of the activity of glycerokinase was performed at the same conditions. The current change was monitored kinetically and activity was expressed in μπιοΐ of ADP forming by glycerokinase per 1 min. The measured activities of glycerokinase preparations (12.3 U/mg and 475 U/ml, respectively) agreed with th declared by the supplier (11.9 U/mg and 493 U/ml, respectively).

Example 5

A concentration of free fatty acids (substrate of AMP producing enzyme) was determined by a fluorometric measurement. The reaction mixture (0.45 ml) contained the reagents in 0.1 M Tris buffer (pH 7.8) as follows: 0.1 U of acyl-CoA-synthetase (Sigma, St. Louis, USA, Cat. No. A 3352), 1 U of myokinase (Sigma, St. Louis, USA, Cat. No. M 3003), 0.5 U of creatine kinase, 0.2 U of creatinase, 0.4 U of sarcosine oxidase, 1.5 U of peroxidase, 0.5 mM coenzyme A trilithium salt (Sigma, St. Louis, USA, Cat. No. C 3019), 2 mM ATP, 2 mM creatine phosphate, 2 g/1 Triton X-100, 2 g/1 sodium taurocholate (Sigma, St. Louis, USA, Cat. No. T 0750), 1 mM europium (III) tetracycline complex (obtained by mixing aqueous solutions of europium chloride and tetracycline hydrochloride), 2 mM magnesium chloride. Oleate solutions (50 μΐ) of various concentrations were added to this reaction mixture. After 1 min the fluorescent intensity was measured by fluorometer at the wavelength of 615 nm using the excitation wavelength of 400 nm. The calibration curve for oleate is illustrated in Fig. 4.

Example 6

A concentration of hexoses (kinase substrate) was determined by an multienzyme biosensor. A palladium electrode (2 mm diameter; Amel, Milan, Italy) was cleaned and covered with an film of polyvinyalcohol. A solution of enzymes was prepared by mixing hexokinase (10 mg/ml, Sigma, St. Louis, USA, Cat. No. H 6380), creatine kinase (8 mg/ml), creatinase (12 mg/ml), sarcosine oxidase (35 mg/ml ), and glutaraldehyde (0.5 mg/ml). 2 μΐ of this solution were placed into the surface of the electrode and left to dry for 3 h at room temperature followed by 15 min at 40°C. The biosensor was immersed in 10 ml of phosphate buffer (pH 7.3) containing. 10 mM ATP, 5 mM creatine phosphate, 2 mM magnesium acetate, 4 mM ferricyanide and a current was monitored at the constant potential of 300 mV against a reference electrode (SCE). The biosensor was calibrated by successive additions of the equimolar solution of glucose and fructose. The calibration curve for hexoses is illustrated in Fig. 5.

Example 7

A concentration of caffeine (inhibitor of AMP producing enzyme) was determined by an multienzyme biosensor. The planar carbon screen printed electrode (1.6 mm diameter, prepared using carbon printing ink of Gwent Electronic Materials, Ltd., UK, Cat. No. C2000802D2) was cleaned and modified with ferrocene by adsorption. A solution of enzymes with an activator was prepared by mixing cyclic-3', 5 '-nucleotide phosphodiesterase (10 mg/ml, Sigma, St. Louis, USA, Cat. No. P 0520), calmodulin (12,500 U/ml, Sigma, St. Louis, USA, Cat. No. P 0520), myokinase (5 mg/ml), creatine kinase (6 mg/ml), creatinase (9 mg/ml), sarcosine oxidase (30 mg/ml ), peroxidase (5 mg/ml), and glutaraldehyde (0.5 mg/ml). 1 μΐ of this solution was placed into the surface of the electrode and left to dry for 3 h at room temperature followed by 15 min at 35 °C. The biosensor was immersed in 10 ml of Tris buffer (pH 7.5) containing 3 mM ATP, 3 mM creatine phosphate, 2 mM magnesium acetate, 10 mM calcium chloride, 1 mM cAMP (Sigma, St. Louis, USA, Cat. No. A 9501), 2 mM ferrocyanide. The constant potential of 0 mV against a reference electrode (SCE) was applied and the initial current was monitored after 1 min of the a signal stabilisation. The initial current corresponded to the maximum phosphodiesterase activity. After additions of caffeine, which inhibited phosphodiesterase, the current decreased proportionally to its concentration. The calibration curve for caffeine is illustrated in Fig. 6.

Claims

1. A method of quantitative determining adenosine diphosphate comprising:

a) a phosphorylation of adenosine- diphosphate with creatine kinase and creatine phosphate to form creatine;

b) a transformation of said creatine with creatinase to sarcosine;

c) an oxidation of said sarcosine with sarcosine oxidase in the presence of an electron- acceptor;

d) a measurement of a decrease of said electron-acceptor or a measurement of an increase of said electron-acceptor or an increase of hydrogen peroxide, where said decrease or said increase is proportional to the amount of adenosine diphosphate;

wherein the reactions are performed at a temperature from 5 °C to 50 °C and pH from 5.0 to 9.5.

2. The method of quantitative determining adenosine diphosphate of claim 1, where said electron-acceptor is oxygen.

3. The method of quantitative determining adenosine diphosphate of claim 1, where said electron-acceptor is selected from the group of methylene blue, thionine, neutral red, toluidine blue, Meldola's blue, ferricyanide, ferricinium and its derivates, resorufin derivates, rhodamines, coumarin derivates.

4. The method of quantitative determining adenosine diphosphate of claims lto 3, where enzyme activators and/or enzyme stabilisers and/or surfactants are added to the reagent comprising creatine kinase, creatine phosphate, creatinase, and sarcosine oxidase.

5. The method of quantitative determining adenosine diphosphate of claim 4, where said enzyme activator is a compound containing Mg²⁺ cation.

6. The method of quantitative determining adenosine diphosphate of claims lto5, where the reagent comprising creatine kinase, creatine phosphate, creatinase, and sarcosine oxidase is in a free form and/or an immobilised form.

7. The method of quantitative determining adenosine diphosphate of claim 6, where said reagent is a biosensor.

8. The method of quantitative determining adenosine diphosphate of claims 1 to7, where the reactions are performed at pH from 6.5 to 8.0.

9. The method of quantitative determining adenosine diphosphate of claims 1 to 8, where said way is used for determining adenosine monophosphate, after a transformation of adenosine monophosphate to adenosine diphosphate with myokinase in the presence of adenosine triphosphate.

10. The method of quantitative determining adenosine diphosphate of claims 1 to 8, where said method is used for determining cyclic adenosine monophosphate, after a hydrolysis of cyclic adenosine monophosphate to adenosine monophosphate with cyclic-3 ',5 '-nucleotide phosphodiesterase and the transformation of adenosine monophosphate to adenosine diphosphate with myokinase in the presence of adenosine triphosphate.

11. The method of quantitative determining adenosine diphosphate of claims 1 to 8, where said method is used for determining enzymatic activity of the enzyme belonging to the group of kinases after a reaction of said kinase with adenosine triphosphate to form adenosine diphosphate.

12. The method of quantitative determining adenosine diphosphate of claims 1-8, where said method is used for determining the compound which is a substrate or an inhibitor of the enzyme belonging to the group of kinases, after a reaction of said kinase with said substrate and adenosine triphosphate to form adenosine diphosphate, in the case the inhibitor is determined in the presence thereof during said reaction.

13. The method of quantitative determining adenosine diphosphate of claims 1-8, where said method is used for determining the compound which is a substrate or an inhibitor of the enzyme catalysing the formation of adenosine monophosphate, after a reaction of said enzyme with said substrate to form adenosine monophosphate and the transformation of adenosine monophosphate to adenosine diphosphate with myokinase in the presence of ATP, in the case the inhibitor is determined in the presence thereof during said reaction.