By a News Reporter-Staff News Editor at Life Science Weekly — From Alexandria, Virginia, NewsRx journalists report that a patent by the inventors Hart, Russell (Chelsea, MI); Scheuer, Barbara (Chelsea, MI); Trievel, Raymond (Ypsilanti, MI), filed on September 10, 2013, was published online on July 1, 2014 (see also Arbor Assays LLC).
The patent’s assignee for patent number 8765396 is Arbor Assays LLC (Ann Arbor, MI).
News editors obtained the following quote from the background information supplied by the inventors: “Formaldehyde is common byproduct formed in the oxidative demethylation of proteins, nucleic acids, and biological small molecules
“Conventional methods for measuring enzyme-mediated oxidative demethylation are limited and cannot be used in high throughput screening for modulators of the enzymes. For example, known assays for formaldehyde formation from a demethylase reaction involve the use of radiolabeled (.sup.14C or .sup.3H) methyl-peptide substrates with the subsequent formation, isolation and quantitation of radioactive formaldehyde. Alternative methods have included measurement of the formation of hydrogen peroxide as a byproduct of enzyme activity or Western blotting using methyl lysine specific antibodies. The measurement of hydrogen peroxide product to determine the activity of oxidative demethylases can be problematic in drug screening scenarios since the peroxide formed can react with the test compound or with other components of the demethylase reaction.
“Despite their biological importance, oxidative demethylase enzyme activities have proven difficult to quantitatively assay hindering understanding of their kinetic properties, substrate specificities, and reaction mechanisms.
“Thus, there is a continuing need for methods and compositions for detection of formaldehyde byproducts of enzyme activity and sensitive assays for enzyme activity.”
As a supplement to the background information on this patent, NewsRx correspondents also obtained the inventors’ summary information for this patent: “Methods of assaying enzyme-mediated oxidative demethylation are provided according to embodiments of the present invention which includes combining, under reaction conditions, an oxidative demethylation enzyme, a substrate for the oxidative demethylation enzyme and a formaldehyde detection reagent. Detection of fluorescence is indicative of formaldehyde generated by oxidative demethylation of the substrate by the enzyme, the fluorescence resulting from reaction of formaldehyde and the formaldehyde detection reagent.
“In particular embodiments of methods of the present invention, an oxidative demethylation enzyme activity detected is a histone demethylase activity, a DNA demethylase activity or a P450 demethylase enzyme activity. In a preferred option, the oxidative demethylation enzyme activity assayed is a human oxidative demethylation enzyme.
“In embodiments of the present invention, the formaldehyde detection reagent includes 4-amino-3-penten-2-one.
“Embodiments of the present invention include mixing ammonium acetate, acetyl acetone and acetic acid in a solvent to produce the formaldehyde detection reagent. The ammonium acetate added to a concentration in the range of about 0.125M-8M, inclusive, the acetyl acetone added to a concentration in the range of about 0.005M-1.6M, inclusive, and the acetic acid added to a concentration in the range of about 0.0125M-3.2M, inclusive. In preferred embodiments, the formaldehyde detection reagent so produced is not purified to remove unreacted ammonium acetate, acetyl acetone and/or acetic acid.
“Method of producing a formaldehyde detection reagent are provided according to the present invention which include mixing ammonium acetate, acetyl acetone and acetic acid in a solvent, the ammonium acetate added to a concentration in the range of about 0.125M-8M, inclusive, the acetyl acetone added to a concentration in the range of about 0.005M-1.6M, inclusive, and the acetic acid added to a concentration in the range of about 0.0125M-3.2M, inclusive, to produce the formaldehyde detection reagent. Optionally, the solvent is aqueous.
“Methods of screening a test agent for activity to modulate an oxidative demethylation enzyme are provided according to embodiments of the present invention which include preparing a test reaction including an oxidative demethylation enzyme, a substrate for the oxidative demethylation enzyme, a test agent and a formaldehyde detection reagent under reaction conditions which allow generation of formaldehyde by oxidative demethylation of the substrate by the enzyme in the absence of the test agent. Fluorescence is assayed in the test reaction and detected fluorescence is indicative of reaction of formaldehyde and the formaldehyde detection reagent in the test reaction. Assays according to embodiments of the present invention allow precise quantitation of formaldehyde and therefore inventive assays allow a user to determine enzyme kinetic characteristics for each particular oxidative demethylase enzyme, for example in the presence or absence of a putative modulator.
“Fluorescence detected in the test reaction is compared with fluorescence detected in a control and a difference in detected fluorescence comparing the test reaction and control is indicative of a change in activity of the oxidative demethylation enzyme in the presence of the test agent.
“Optionally, a plurality of test reactions is prepared, fluorescence is detected in the plurality of test reactions and the detected fluorescence in the plurality of test reactions is compared with one or more controls to determine whether a test agent is a modulator of demethylating activity of an oxidative demethylation enzyme.
“Methods of detecting formaldehyde are provided according to embodiments of the present invention which includes reacting a formaldehyde detection reagent and a sample to be tested for the presence of formaldehyde, thereby generating a test sample; and detecting fluorescence in the test sample. In preferred embodiments the sample is a liquid sample and the formaldehyde detection reagent is a liquid.
“Methods of detecting formaldehyde are provided according to embodiments of the present invention which include mixing ammonium acetate, acetyl acetone and acetic acid in a solvent, the ammonium acetate added to a concentration in the range of about 0.125M-8M, inclusive, the acetyl acetone added to a concentration in the range of about 0.005M-1.6M, inclusive, and the acetic acid added to a concentration in the range of about 0.0125M-3.2M, inclusive, to produce a formaldehyde detection reagent; reacting the formaldehyde detection reagent with a sample to be tested for the presence of formaldehyde, thereby generating a test sample; and detecting fluorescence in the test sample. In preferred embodiments, the formaldehyde detection reagent so produced is not purified to remove unreacted ammonium acetate, acetyl acetone and/or acetic acid prior to reaction with the sample. Thus, for example, following combination of ammonium acetate, acetyl acetone and acetic acid in a solvent to produce a solution which is a formaldehyde detection reagent, also called Luminos reagent herein, in preferred embodiments the formaldehyde detection reagent is used ‘as is’ without purification, addition, or removal of material from the solution. Optionally, in preferred embodiments, a preservative, such as an antimicrobial, is added to the Luminos reagent and the formaldehyde detection reagent including the preservative is used ‘as is’ without purification, addition, or removal of material from the solution.
“Kits for detecting liquid phase formaldehyde are provided according to embodiments of the present invention which include a formaldehyde detection reagent, the reagent produced by mixing ammonium acetate, acetyl acetone and acetic acid in a solvent, the ammonium acetate added to a concentration in the range of about 0.125M-8M, inclusive, the acetyl acetone added to a concentration in the range of about 0.005M-1.6M, inclusive, and the acetic acid added to a concentration in the range of about 0.0125M-3.2M, inclusive. In preferred embodiments, the formaldehyde detection reagent so produced is not purified to remove unreacted ammonium acetate, acetyl acetone and/or acetic acid prior to reaction with the sample.”
For additional information on this patent, see: Hart, Russell; Scheuer, Barbara; Trievel, Raymond. Method of Screening a Test Agent for Activity to Modulate an Oxidative Demethylation Enzyme. U.S. Patent Number 8765396, filed September 10, 2013, and published online on July 1, 2014. Patent URL: http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO1&Sect2=HITOFF&d=PALL&p=1&u=\%2Fnetahtml\%2FPTO\%2Fsrchnum.htm&r=1&f=G&l=50&s1=8765396.PN.&OS=PN/8765396RS=PN/8765396
Keywords for this news article include: Chemicals, Chemistry, Acetic Acids, Formaldehyde, Acyclic Acids, Arbor Assays LLC, Hydrogen Peroxide, Enzymes and Coenzymes.
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