Methane → Phosphate
Reaction equation
[P:1]([O:13]C[C@H]1O[C@@H](N2C3N=C(N)NC(=O)C=3N=C2)C[C@@H]1O)([O:4][P:5]([O:8][P:9]([OH:12])([OH:11])=[O:10])([OH:7])=[O:6])(=[O:3])[OH:2].C.NCC(O)=O.[Mg+2].[Cl-].[Cl-]>>[O-:12][P:9]([O:8][P:5]([O:4][P:1]([O-:13])([O-:3])=[O:2])([O-:7])=[O:6])([O-:11])=[O:10].[P:1]([O-:13])([O-:4])([O-:3])=[O:2]Participants
Procedure
Enzyme assay procedures were performed in a manner similar to those described by Seto et al.(13) by measuring the hydrolysis of dGTP to Norit non-adsorbable PPPi. The incubation mixture had a volume of 55 μL and included 2.0 mM dGTP; 67 mM Glycine, pH 8.5; 6.7 mM MgCl2; and 0.02-0.2 milliunit of enzyme. After 20 min at 37° C., the reaction was terminated by addition of an acidic suspension of Norit A. The mixture was centrifuged, and an aliquot of the supernatant was brought to 0.15 N HCl, and was boiled for 15 minutes. This step in the procedure resulted in the hydrolysis of tripolyphosphate to orthophosphate. The total orthophosphate concentration was determined colorimetrically by the addition of an ascorbate-molybdate solution. The absorbance at 780 nm was determined after incubating the mixture at 45° C. for 15 minutes. Results were compared to a phosphate standard curve. A unit of enzyme forms 1 μmol of tripolyphosphate under these conditions. The stimulatory effect of ssDNA was assayed by adding 50 μg/mL of m 13 mp 18 DNA to the standard assay.
Workup
- customthe reaction was terminated by addition of an acidic suspension of Norit A
- waitwas boiled for 15 minutes
Sources & evidence
- ORD record
- ord-accee7211e754126bce3a536b9183685
- Source dataset
- uspto-grants-2004_02
- Patent number
- US06696254B2
Reaction notes
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