# US 2014/0273036 A1 · Luminescent detection of inorganic phosphate and coupled reactions > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2014/0273036 A1 - Document ID: US20140273036A1 - Status: Published application - Kind: A1 - Application number: 14/205,909 - Filed: 2014-03-12 - Published: 2014-09-18 - Earliest claimed: 2013-03-12 - Assignees: Promega Corporation - Applicants: Promega Corporation - Inventors: Subhanjan Mondal; Kuei-Hsuan Hsiao; Said Goueli - Web page: https://patentgrep.com/patent/US20140273036A1 ## CPC - C12Q 1/66: involving luciferase - G01N 33/84: involving inorganic compounds or pH - G01N 2333/91265: Polyribonucleotide nucleotidyl transferases, i.e. polynucleotide phosphorylase (2.7.7.8) - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## Abstract Luminescent detection of inorganic phosphate is carried out in an assay through the intermediary enzymatic production of ADP. ADP is converted to ATP which is used in a luminescent reaction. The assay can be used to monitor coupled enzyme reactions which use or generate inorganic phosphate and the modulation of such reactions. ## Claims ### Claim 1 A method for detecting the presence or absence of or determining the amount of inorganic phosphate (Pi) in a solution, the method comprising: (a) contacting the solution with polynucleotide phosphorylase to convert the inorganic phosphate to ADP; (b) converting the ADP produced from the inorganic phosphate to ATP with an enzyme that catalyzes the conversion of ADP to ATP; and (c) detecting the ATP produced from the ADP using a luminescent reaction. ### Claim 2 (depends on 1) The method of claim 1, wherein the conversion of ADP to ATP occurs concurrently with the conversion of inorganic phosphate to ADP. ### Claim 3 (depends on 1) The method of claim 1, wherein the luminescent reaction occurs concurrently with the conversion of ADP to ATP. ### Claim 4 (depends on 1) The method of claim 1, wherein steps (a) (b) and (c) are carried out as a homogeneous assay. ### Claim 5 (depends on 1) The method of claim 1, wherein steps (a) (b) and (c) are performed in a single reaction vessel. ### Claim 6 (depends on 1) The method of claim 1, wherein the solution comprises a crude cell lysate. ### Claim 7 (depends on 1) The method of claim 1, further comprising generating inorganic phosphate in the solution with an inorganic phosphate-generating enzyme, and wherein the luminescent reaction provides a measure of the activity of the inorganic phosphate-generating enzyme. ### Claim 8 (depends on 7) The method of claim 7, further comprising modulating the activity of the inorganic phosphate-generating enzyme with a composition comprising a modulator, wherein the luminescent reaction provides a measure of the effect of the modulator on the activity of the inorganic phosphate-generating enzyme. ### Claim 9 (depends on 7) The method of claim 7, wherein the measure is quantitative. ### Claim 10 (depends on 7) The method of claim 7, wherein the generation of inorganic phosphate occurs concurrently with the conversion of inorganic phosphate to ADP. ### Claim 11 (depends on 7) The method of claim 7, wherein the generation of inorganic phosphate occurs prior to the conversion of inorganic phosphate to ADP. ### Claim 12 (depends on 7) The method of claim 7, wherein the inorganic phosphate-generating enzyme is a lipid phosphatase, protein phosphatase, inositol phosphatase, transcarbamylase, phosphorylase, nucleotidase, ATPase, GTPase or tubulin. ### Claim 13 (depends on 7) The method of claim 7, wherein the inorganic phosphate-generating enzyme is a pyrophosphatase and further comprising generating inorganic pyrophosphate in the solution with an inorganic-pyrophosphate-generating enzyme, and wherein the luminescent reaction provides a measure of the activity of the inorganic pyrophosphate-generating enzyme. ### Claim 14 (depends on 1) The method of claim 1 wherein the enzyme that catalyzes the conversion of the ADP to ATP is a pyruvate kinase. ### Claim 15 (depends on 1) The method of claim 1 wherein the luminescent reaction comprises a luciferase enzyme and a substrate for the luciferase enzyme. ### Claim 16 (depends on 15) The method of claim 15, wherein the luciferase enzyme is a recombinant luciferase enzyme. ### Claim 17 (depends on 15) The method of claim 15, wherein the luciferase enzyme is a thermostable luciferase enzyme, chemostable luciferase enzyme, or a combination thereof. ### Claim 18 A kit for detecting inorganic phosphate in a solution, the kit comprising: (i) a polynucleotide phosphorylase; (ii) poly(A) (iii) an enzyme that catalyzes the conversion of ADP to ATP; (iv) a luciferase enzyme; and (v) a substrate for the luciferase enzyme. ### Claim 19 (depends on 18) The kit of claim 18 further comprising one or more of: (vi) a phosphate group donor; (vii) one or more detergents; (viii) one or more buffer solutions; and/or (ix) one or more salts. ### Claim 20 (depends on 19) The kit of claim 19, comprising a phosphate group donor, wherein the enzyme that catalyzes the conversion of the ADP to ATP is a pyruvate kinase, and the phosphate group donor is phosphoenolpyruvate. ### Claim 21 (depends on 18) The kit of claim 18, wherein a first composition comprises (i) and (ii) and a different composition comprises (iii), (iv) and (v). ### Claim 22 (depends on 18) The kit of claim 18, wherein the kit further comprises pyrophosphatase.