WO2000042214A1 - Phosphate release enzyme assay - Google Patents
Phosphate release enzyme assay Download PDFInfo
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- WO2000042214A1 WO2000042214A1 PCT/GB2000/000088 GB0000088W WO0042214A1 WO 2000042214 A1 WO2000042214 A1 WO 2000042214A1 GB 0000088 W GB0000088 W GB 0000088W WO 0042214 A1 WO0042214 A1 WO 0042214A1
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- phosphate
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- pyrophosphate
- shikimate
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- 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
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/42—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase
Definitions
- the present invention relates to assays for the determination of an inhibitory effect of a compound on an enzyme.
- the present invention relates to the determination of an inhibitory effect on any one of a number of enzymes in an enzyme-coupled system.
- target systems that are desirable to control comprise enzymes. If an enzyme itself is the target there is usually the requirement for its isolation and purification prior to assay in the presence of chemical compounds. In many cases, the enzyme is only one of a number involved in a particular biological pathway. The aim of a drug development programme may ultimately be the controlled inhibition of the pathway achieved through inhibition of any one (or several) of a number of enzymes common to the pathway. Screening for activity in these pathways is difficult, since there are different reactants and products for each enzyme; consequently, it is more usual to isolate a single enzyme and screen for activity against it, normally using a measurement of product formation as the basis for the screen.
- a method for determining an inhibitory effect of a compound on an enzyme comprises the step of determining the difference in the amount of phosphate released in the absence and presence of the compound; in particular, the assay is conducted when the enzyme is present in a system comprising two or more enzyme-coupled reactions, wherein phosphate is generated as a final product of the system.
- the method comprises the addition of pyrophosphatase to an enzyme- coupled reaction producing PPi, and measuring the amount of phosphate generated. This can then be used to quantify how much pyrophosphate was generated in the system.
- the phosphate may be measured by the addition of a substrate that reacts with phosphate to generate a chromophoric substance.
- the phosphate-reactive substrate may be 2 -amino-6-mercapto-7 -methylaminopur ine ribonucleoside.
- the enzyme that is inhibited in the system may be identified by the separate addition of the substrates for each of the enzymes present in the system and subsequently determining the amount of phosphate generated. If an increase in phosphate is detected, this indicates that the substrate acted downstream of the inhibited enzyme and this therefore helps to identify the particular enzyme that has been inhibited.
- the present invention therefore provides a method for assaying different enzymes in a pathway in an efficient manner, whereby inhibition of any one enzyme is reflected in the downstream inhibition of phosphate or PPi production by the final enzyme.
- Figure 1 illustrates the co-enzyme A biosynthetic pathway and the measurement of phosphate
- Figure 2 illustrates the folate biosynthetic pathway and the measurement of the final product, phosphate
- FIG. 3 illustrates the shiki ate pathway and the measurement of the final product, phosphate.
- a major advantage of the present method is that it allows two or more different enzymes to be screened together thereby reducing the necessity for separate assay screens for each enzyme. Chemicals of interest can then be used in the screens and an inhibition event detected. This allows the easy identification of chemical compounds that may have interesting inhibitory properties. The specific target enzyme may then be identified in later experiments for further study. The skilled person will appreciate how to couple enzyme reactions to achieve sequential enzymatic reactions resulting in phosphate (or pyrophosphate) release. The coupled system does not have to be one found in nature, but may be apparent due to a shared product/substrate between two enzymes.
- Coenzyme A CoA synthesising enzymes: Pantothenate kinase (PtK) and 4-phosphopantetheine adenylyltransferase (PPAT) .
- PtK Pantothenate kinase
- PPAT 4-phosphopantetheine adenylyltransferase
- the two enzymes PtK and PPAT catalyse non-sequential reactions in the pathway of CoA biogenesis in vivo .
- the reaction catalysed by PtK involves the transfer of the ⁇ phosphate of ATP to pantothenate, yielding 4- phosphopantothenate.
- PPAT is the transfer of adenosine monophosphate (AMP) moiety of ATP to 4-phosphopantetheine, generating dephosphocoenzyme A (dCoA) and PPi.
- AMP adenosine monophosphate
- PPAT can be linked in an assay sequence which culminates in PPi release, the latter may then (optionally) be cleaved to release phosphate.
- the production of phosphate or PPi in the final enzyme step may be measured by spectrophotometric means. Any increase in absorbance at 360 nm is dependent on ATP, PtK, PPAT and pantetheine (or suitable analog or derivative) and indicates that the combined activities of PtK and PPAT are being measured.
- the addition of an inhibitor of PtK will result in a decrease in the measured production of phosphate due to the lack of substrate production for PPAT.
- the reduction in the rate of phosphate production may also indicate an inhibitory effect on PPAT.
- Example A reaction was carried out to measure phosphate release from the enzyme-coupled system, in the presence and absence of inhibitors previously shown to be specific for either PtK or PPAT.
- the reaction assay comprised 0.02 units of PtK and 0.076 units of PPAT (where 1 unit is sufficient to catalyse l ⁇ M of substrate per minute) .
- the substrate for PtK was the pantetheine derivative S - [ 3 ' ( N - propyl) succinamidyl] pantetheine (NPS-pantetheine) (60 ⁇ M), and ATP was also present in the reaction mix at a concentration of ImM.
- Phosphate release was measured using the spectrophotometric assay as disclosed in Webb, supra .
- the compound [N-acetyl (O-t-butyl) D-serinyl] ⁇ -alanyl- 3 , 3-dimethyl-l-butamine was used as the inhibitor of PtK, and 2 , 4 , 3 ' , 5 ' -tetrachlorobenzanilide was used as the inhibitor of PPAT (both at 50 ⁇ m) .
- Inhibition of PtK The rate of phosphate release in the absence of inhibitor was 1.35 ⁇ m/min/ml. In the presence of inhibitor the rate was determined as 0.22 ⁇ m/min/ml, giving an effective rate of inhibition of approximately 80%.
- Inhibition of PPAT The rate of phosphate release in the absence of inhibitor was measured as 0.735 ⁇ m/min/ml. In the presence of inhibitor, the rate was 0.500 ⁇ m/min/ml, giving an inhibition rate of approximately 30%.
- DHNA Dihydroneopterin aldolase
- HPPK hydroxymethyl dihydropterin pyrophosphokinase
- DHPS dihydropteroate synthase
- Dihydroneopterin (IV) is cleaved by DHNA to generate glycoaldehyde (V) and hydroxymethyldihydroneopterin (VI) .
- HPPK then catalyses the attack of the hydroxyl oxygen of hydroxymethyl dihydropterin on the ⁇ -phosphorous atom of ATP to generate hydroxymethyl dihydropetrin pyrophosphate (VII) and AMP as a byproduct.
- PPi is then displaced from dihydroxymethyl dihydropterin pyrophosphate by para-aminobenzoic acid (VIII) in a reaction catalysed by DHPS, the final enzyme in the folate biosynthetic sequence, generating dihydropteroate (IX) .
- the PPi generated by this sequence of three enzymes may be measured, for example using the inorganic pyrophosphatase/ pur ine nucleoside pyrophosphorylase/methylthioguanosine detection system to generate an increase in absorbance at 360 nm.
- the time course of product (PPi) release is linear for at least 10 minutes and is dependent upon the presence of each component of the system.
- HPPK inhibitor ⁇ - ⁇ -methylene adenosine triphosphate
- DHNA ⁇ - ⁇ -methylene adenosine triphosphate
- Dehydroquinate (X) is first converted into dehydroshikimate (XI) by dehydroquinate dehydratase.
- Shikimate dehydrogenase then converts the dehydroshikimate to Shikimate (XII)
- shikimate kinase then catalyses the formation of Shikimate 3-phosphate (XIII) .
- This compound is then catalysed by 5-enolpyruvoyl Shikimate 3-phosphate synthase into 5-enoylpyruvoyl shikimate 3-phosphate (XX) , with the additional conversion of phosphoenol pyruvate (IXX) into phosphate.
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Abstract
A method for determining an inhibitory effect of a compound on an enzyme, where the enzyme is one of two or more in an enzyme-catalysed cascade generating pyrophosphate or phosphate as a final product. The method requires the determination of the difference in the amount of pyrophosphate or phosphate generated in the enzyme cascade in the absence and presence of the target compound.
Description
PHOSPHATE RELEASE ENZYME ASSAY
Field of the Invention
The present invention relates to assays for the determination of an inhibitory effect of a compound on an enzyme. In particular, the present invention relates to the determination of an inhibitory effect on any one of a number of enzymes in an enzyme-coupled system. Background to the Invention
The ability to screen compounds for inhibitory effects on a particular biological target is an increasingly important requirement in the pharmaceutical industry. The availability of many thousands of compounds and the possibility of synthesising many thousands more by combinatorial synthesis techniques have meant that the screening of these compounds for activity is both costly and time-consuming. For example, in a drug discovery programme, there may be much known about the biological system under study, including the activity of the particular target which needs to be controlled; however, it may be necessary to screen many hundreds, possibly thousands, of compounds to identify one with the requisite level of activity. It is therefore important that the screening techniques used can be carried out efficiently without the necessity for complicated or lengthy procedures.
Many target systems that are desirable to control comprise enzymes. If an enzyme itself is the target there is usually the requirement for its isolation and purification prior to assay in the presence of chemical compounds. In many cases, the enzyme is only one of a number involved in a particular biological pathway. The aim of a drug development programme may ultimately be the controlled inhibition of the pathway achieved through inhibition of any one (or several) of a number of enzymes common to the pathway. Screening for activity in these pathways is difficult, since there are different reactants and products for each enzyme; consequently, it is more
usual to isolate a single enzyme and screen for activity against it, normally using a measurement of product formation as the basis for the screen.
In Webb, Proc. Nat. Acad. Sci. (USA), 1992; 89:4884- 4887, there is described an assay for the measurement of phosphate using the chromogenic substrate 2-amino-6- mercapto-7-methylaminopurine ribonucleoside (methyl- thioguanosine) . Phosphate was found to cleave this nucleoside in a reaction catalysed by purine nucleoside phosphorylase, generating ribose l-phosphate and the corresponding free base, 2-amino-6-mercapto-7- methylaminopurine (methylthioguanine; MTG) . Conversion of the nucleoside to MTG generates a readily monitored spectrophotometric signal at 360 n . This assay was later extended by Lloyd et al , Nucleic Acids Research, 1995; 23:2886-2892, to measure inorganic pyrophosphate (PPi) by incorporating inorganic pyrophosphatase in the assay to generate inorganic phosphate (Pi) which can then be assayed as described above. This assay has been used previously in the measurement of activity of isolated enzymes. Summary of the Invention
According to the present invention, a method for determining an inhibitory effect of a compound on an enzyme, comprises the step of determining the difference in the amount of phosphate released in the absence and presence of the compound; in particular, the assay is conducted when the enzyme is present in a system comprising two or more enzyme-coupled reactions, wherein phosphate is generated as a final product of the system.
According to one aspect of the invention, the method comprises the addition of pyrophosphatase to an enzyme- coupled reaction producing PPi, and measuring the amount of phosphate generated. This can then be used to quantify how much pyrophosphate was generated in the system. The phosphate may be measured by the addition of a substrate that reacts with phosphate to generate a chromophoric
substance. For example, the phosphate-reactive substrate may be 2 -amino-6-mercapto-7 -methylaminopur ine ribonucleoside.
According to a further aspect of the invention, if inhibition is detected, the enzyme that is inhibited in the system may be identified by the separate addition of the substrates for each of the enzymes present in the system and subsequently determining the amount of phosphate generated. If an increase in phosphate is detected, this indicates that the substrate acted downstream of the inhibited enzyme and this therefore helps to identify the particular enzyme that has been inhibited.
The present invention therefore provides a method for assaying different enzymes in a pathway in an efficient manner, whereby inhibition of any one enzyme is reflected in the downstream inhibition of phosphate or PPi production by the final enzyme. Description of the Drawings
Figure 1 illustrates the co-enzyme A biosynthetic pathway and the measurement of phosphate;
Figure 2 illustrates the folate biosynthetic pathway and the measurement of the final product, phosphate; and
Figure 3 illustrates the shiki ate pathway and the measurement of the final product, phosphate. Description of the Invention
A major advantage of the present method is that it allows two or more different enzymes to be screened together thereby reducing the necessity for separate assay screens for each enzyme. Chemicals of interest can then be used in the screens and an inhibition event detected. This allows the easy identification of chemical compounds that may have interesting inhibitory properties. The specific target enzyme may then be identified in later experiments for further study. The skilled person will appreciate how to couple enzyme reactions to achieve sequential enzymatic reactions resulting in phosphate (or pyrophosphate) release. The
coupled system does not have to be one found in nature, but may be apparent due to a shared product/substrate between two enzymes.
A description of three specific enzyme coupled systems follows, each of which illustrate the invention with reference to the accompanying drawings.
(1) Coenzyme A (CoA) synthesising enzymes: Pantothenate kinase (PtK) and 4-phosphopantetheine adenylyltransferase (PPAT) . The two enzymes PtK and PPAT catalyse non-sequential reactions in the pathway of CoA biogenesis in vivo . The reaction catalysed by PtK involves the transfer of the γ~ phosphate of ATP to pantothenate, yielding 4- phosphopantothenate. The reaction catalysed by PPAT is the transfer of adenosine monophosphate (AMP) moiety of ATP to 4-phosphopantetheine, generating dephosphocoenzyme A (dCoA) and PPi. Clearly, no product of either enzyme reaction forms the substrate of the other. However, PtK is able to phosphorylate pantetheine (I) (or other suitable analogs or derivatives) as efficiently as pantothenate, yielding 4- phosphopantetheine (II) rather than 4-phosphopantothenate. 4-phosphopantetheine is a substrate for PPAT which, on addition of ATP, produces dCoA (III) and PPi (see Figure 1) . Therefore, although the reactions catalysed by PtK and PPAT are not sequential in vivo , the activities of PtK and
PPAT can be linked in an assay sequence which culminates in PPi release, the latter may then (optionally) be cleaved to release phosphate.
The production of phosphate or PPi in the final enzyme step may be measured by spectrophotometric means. Any increase in absorbance at 360 nm is dependent on ATP, PtK, PPAT and pantetheine (or suitable analog or derivative) and indicates that the combined activities of PtK and PPAT are being measured. The addition of an inhibitor of PtK will result in a decrease in the measured production of phosphate due to the lack of substrate production for PPAT. The reduction in
the rate of phosphate production may also indicate an inhibitory effect on PPAT.
The following Example illustrates this system. Example A reaction was carried out to measure phosphate release from the enzyme-coupled system, in the presence and absence of inhibitors previously shown to be specific for either PtK or PPAT.
The reaction assay comprised 0.02 units of PtK and 0.076 units of PPAT (where 1 unit is sufficient to catalyse lμM of substrate per minute) . The substrate for PtK was the pantetheine derivative S - [ 3 ' ( N - propyl) succinamidyl] pantetheine (NPS-pantetheine) (60μM), and ATP was also present in the reaction mix at a concentration of ImM.
Phosphate release was measured using the spectrophotometric assay as disclosed in Webb, supra .
The compound [N-acetyl (O-t-butyl) D-serinyl] β-alanyl- 3 , 3-dimethyl-l-butamine was used as the inhibitor of PtK, and 2 , 4 , 3 ' , 5 ' -tetrachlorobenzanilide was used as the inhibitor of PPAT (both at 50μm) .
Inhibition of PtK: The rate of phosphate release in the absence of inhibitor was 1.35 μm/min/ml. In the presence of inhibitor the rate was determined as 0.22 μm/min/ml, giving an effective rate of inhibition of approximately 80%.
Inhibition of PPAT: The rate of phosphate release in the absence of inhibitor was measured as 0.735 μm/min/ml. In the presence of inhibitor, the rate was 0.500 μm/min/ml, giving an inhibition rate of approximately 30%.
(2) Folate biosynthetic enzymes: Dihydroneopterin aldolase (DHNA) , hydroxymethyl dihydropterin pyrophosphokinase (HPPK) and dihydropteroate synthase (DHPS) .
The above three enzymes catalyse a naturally-occurring sequence of reactions in the pathway of microbial folate synthesis (illustrated in Figure 2) . Dihydroneopterin (IV) is cleaved by DHNA to generate glycoaldehyde (V) and
hydroxymethyldihydroneopterin (VI) . HPPK then catalyses the attack of the hydroxyl oxygen of hydroxymethyl dihydropterin on the β-phosphorous atom of ATP to generate hydroxymethyl dihydropetrin pyrophosphate (VII) and AMP as a byproduct. PPi is then displaced from dihydroxymethyl dihydropterin pyrophosphate by para-aminobenzoic acid (VIII) in a reaction catalysed by DHPS, the final enzyme in the folate biosynthetic sequence, generating dihydropteroate (IX) . The PPi generated by this sequence of three enzymes may be measured, for example using the inorganic pyrophosphatase/ pur ine nucleoside pyrophosphorylase/methylthioguanosine detection system to generate an increase in absorbance at 360 nm. The time course of product (PPi) release is linear for at least 10 minutes and is dependent upon the presence of each component of the system.
The addition of the HPPK inhibitor, β-γ-methylene adenosine triphosphate, decreases the observed rate of the coupled enzyme reaction. As the inhibitor has no effect on the other enzymes (DHNA or DHPS) , this illustrates that the coupling of the activities of DHNA, HPPK and DHPS so as to generate PPi (and ultimately phosphate) provides a simple and practical means for simultaneous screening of each and every one of the three enzymes for inhibitors of microbial folate synthesis.
(3) The enzymes: dehydroquinate dehydratase, shikimate dehydrogenase, shikimate kinase and enoylpyruvoyl shikimate 3-phosphate synthese.
The above enzymes catalyse steps (shown in Figure 3) in the so-called shikimate pathway that results ultimately in the production of enoylpyruvoylshikimate 3-phosphate.
Dehydroquinate (X) is first converted into dehydroshikimate (XI) by dehydroquinate dehydratase. Shikimate dehydrogenase then converts the dehydroshikimate to Shikimate (XII) , and shikimate kinase then catalyses the formation of Shikimate 3-phosphate (XIII) . This compound is then catalysed by 5-enolpyruvoyl Shikimate 3-phosphate
synthase into 5-enoylpyruvoyl shikimate 3-phosphate (XX) , with the additional conversion of phosphoenol pyruvate (IXX) into phosphate.
Claims
1. A method for determining an inhibitory effect of a compound on an enzyme, wherein the enzyme is one of two or more in an enzyme-coupled cascade generating pyrophosphate or phosphate as a final product, comprising the step of determining the difference in the amount of pyrophosphate or phosphate generated in the enzyme cascade in the absence and presence of the target compound.
2. A method according to claim 1, wherein pyrophosphate is determined by the addition of pyrophosphatase and measuring the amount of phosphate generated.
3. A method according to claim 1 or claim 2, which comprises adding a substrate that reacts with phosphate to generate a chromophoric substance.
4. A method according to claim 3 , wherein the phosphate- reactive substrate is 2 -amino-6-mercapto-7 - methylaminopurine ribonucleoside .
5. A method according to any preceding claim, further comprising the subsequent step of adding a substrate for one of the other enzymes in the cascade and determining any difference in phosphate or pyrophosphate generated in the system.
6. A method according to any preceding claim, wherein the cascade comprises pantothenate kinase and phosphopantetheine adenylyltransferase.
7. A method according to any of claims 1 to 5 , wherein the cascade comprises dihydroneopterin aldolase, hydroxymethyl dihdropterin pyrophosphokinase and dihydropteroate synthase.
8. A method according to any of claims 1 to 5 , wherein the cascade comprises dehydroquinate dehydratase, shikimate dehydrogenase, shikimate kinase and 5-enoylpyruvoyl shikimate 3-phosphate synthase.
9. An enzyme inhibitor identified using a method according to any preceding claim.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU19948/00A AU1994800A (en) | 1999-01-15 | 2000-01-14 | Phosphate release enzyme assay |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB9900931.8 | 1999-01-15 | ||
| GB9900931 | 1999-01-15 |
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| Publication Number | Publication Date |
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| WO2000042214A1 true WO2000042214A1 (en) | 2000-07-20 |
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|---|---|---|---|
| PCT/GB2000/000088 WO2000042214A1 (en) | 1999-01-15 | 2000-01-14 | Phosphate release enzyme assay |
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| WO (1) | WO2000042214A1 (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002016601A2 (en) * | 2000-08-24 | 2002-02-28 | Omnigene Bioproducts, Inc. | Microorganisms and assays for the identification of antibiotics |
| EP1281769A1 (en) * | 2001-08-01 | 2003-02-05 | Aventis Pharma S.A. | Screening-method for inhibitors of folate-synthesis-pathway enzymes |
| EP1467211A1 (en) * | 2003-04-10 | 2004-10-13 | Tecan Trading AG | Method for the measurement of chemical groups bonded to biological molecules |
| US20130065262A1 (en) * | 2010-05-24 | 2013-03-14 | Research And Diagnostic Systems, Inc. | Phosphatase coupled glycosyltransferase assay |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1994023059A2 (en) * | 1993-03-27 | 1994-10-13 | The University Court Of The University Of Dundee | Anti-microbial agents and screening method therefor |
-
2000
- 2000-01-14 AU AU19948/00A patent/AU1994800A/en not_active Abandoned
- 2000-01-14 WO PCT/GB2000/000088 patent/WO2000042214A1/en active Application Filing
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1994023059A2 (en) * | 1993-03-27 | 1994-10-13 | The University Court Of The University Of Dundee | Anti-microbial agents and screening method therefor |
Non-Patent Citations (2)
| Title |
|---|
| LLOYD, A.J. ET AL.: "A broadly applicable continuous spectrophotometric assay for measuring aminoacyl-tRNA synthetase activity", NUCLEIC ACIDS RESEARCH, vol. 23, no. 15, 1995, pages 2886 - 2892, XP002132846 * |
| MARTIN R. WEBB: "A continuous spectrophotometric assay for inorganic phosphate and for measuring phosphate release kinetcis in biological systems.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, USA, vol. 89, June 1992 (1992-06-01), pages 4884 - 4887, XP002132845 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002016601A2 (en) * | 2000-08-24 | 2002-02-28 | Omnigene Bioproducts, Inc. | Microorganisms and assays for the identification of antibiotics |
| WO2002016601A3 (en) * | 2000-08-24 | 2003-01-23 | Omnigene Bioproducts Inc | Microorganisms and assays for the identification of antibiotics |
| EP1281769A1 (en) * | 2001-08-01 | 2003-02-05 | Aventis Pharma S.A. | Screening-method for inhibitors of folate-synthesis-pathway enzymes |
| WO2003012132A2 (en) * | 2001-08-01 | 2003-02-13 | Aventis Pharma S.A. | Screening method for inhibitors of folate synthesis pathway enzymes |
| WO2003012132A3 (en) * | 2001-08-01 | 2003-08-28 | Aventis Pharma Sa | Screening method for inhibitors of folate synthesis pathway enzymes |
| EP1467211A1 (en) * | 2003-04-10 | 2004-10-13 | Tecan Trading AG | Method for the measurement of chemical groups bonded to biological molecules |
| US7223610B2 (en) | 2003-04-10 | 2007-05-29 | Tecan Trading Ag | Direct phosphorylation state monitoring on biomolecules |
| US20130065262A1 (en) * | 2010-05-24 | 2013-03-14 | Research And Diagnostic Systems, Inc. | Phosphatase coupled glycosyltransferase assay |
| US9410182B2 (en) * | 2010-05-24 | 2016-08-09 | Bio-Techne Corporation | Phosphatase coupled glycosyltransferase assay |
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| Publication number | Publication date |
|---|---|
| AU1994800A (en) | 2000-08-01 |
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