Kinetic Characterization of Histone Acetylation

Steady-state and single turnover kinetics for H3 and H3K14ac were performed under identical buffer conditions (100 mM HEPES buffer (pH 6.8) and 0.08% Triton X-100) at 37°C. Histone H3 concentrations were determined by the measurements of OD₂₇₆ (ε₂₇₆ = 4040). Steady-state (E<>S) assays contained saturating 3 µM Gcn5, 0.5 µM H3 and 200 µM acetyl-CoA. At varying time points, assays were quenched/precipitated with 25% 4°C TCA, and the precipitate was then washed twice with 150 μL acetone (−20°C)[23] (link). Samples were dried, 1.5 μL propionic anhydride was added, and ammonium hydroxide was used to quickly adjust the pH to ∼8 [24] (link). Samples were then incubated at 51°C for 1 h followed by trypsin digestion (overnight at 37°C). In addition, nonenzymatic experiments [25] (link)were conducted under the aforementioned assay procedures in the presence and absence of Gcn5, with 12 µM histone H3 and 100–300 µM acetyl-CoA.

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Kuo Y.M, & Andrews A.J. (2013). Quantitating the Specificity and Selectivity of Gcn5-Mediated Acetylation of Histone H3. PLoS ONE, 8(2), e54896.

Publication 2013

Acetone Acetyl coa Ammonium hydroxide Assay Buffer Digestion Hepes Histone h3 Kinetics Propionic anhydride Triton x 100 Trypsin

Corresponding Organization :

Other organizations : Fox Chase Cancer Center

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Variable analysis

independent variables

Gcn5 concentrations (0.02 to 0.18 µM for steady-state assays)
H3 or H3K14ac concentrations (0.15–45 µM for steady-state assays, 0.5 µM for single turnover assays)
Acetyl-CoA concentrations (0.1–200 µM for steady-state assays, 200 µM for single turnover assays)

dependent variables

Acetylation of H3 and H3K14ac

control variables

Buffer conditions (100 mM HEPES buffer (pH 6.8) and 0.08% Triton X-100)
Temperature (37°C)
Histone H3 concentration measurement (OD276 with ε276 = 4040)
Quenching/precipitation method (25% 4°C TCA, acetone wash)
Derivatization (propionic anhydride, ammonium hydroxide)
Trypsin digestion (overnight at 37°C)

positive controls

Nonenzymatic experiments with 12 µM histone H3 and 100–300 µM acetyl-CoA, in the presence of Gcn5

negative controls

Nonenzymatic experiments with 12 µM histone H3 and 100–300 µM acetyl-CoA, in the absence of Gcn5

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