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  • Characterization of chemical and enzymatic acid-labile phosphorylation of histone H4 using phosphorus-31 nuclear magnetic resonance.

Characterization of chemical and enzymatic acid-labile phosphorylation of histone H4 using phosphorus-31 nuclear magnetic resonance.

Biochemistry (1981-06-09)
J M Fujitaki, G Fung, E Y Oh, R A Smith
ABSTRACT

Phosphorus-31 nuclear magnetic resonance (31P NMR) is used to investigate acid-labile phosphorylation of histone H4. 31P NMR detects phosphorylated histidine residues in in vitro enzymatically phosphorylated H4. The source of kinase is nuclei from either regenerating rat liver or Walker-256 carcinosarcoma. When regenerating rat liver is the source, 31P NMR spectroscopy on the denatured phosphorylated protein exhibits a resonance at 5.3 ppm relative to an 85% orthophosphoric acid external reference. This peak corresponds well with the chemical shift of standard pi-phosphohistidine scanned under similar conditions. Sodium dodecyl sulfate (NaDodSO4)--polyacrylamide gel electrophoresis confirms acid lability. When the source of kinase is Walker-256 carcinosarcoma, the 31P NMR spectrum contains a resonance at 4.9 ppm which corresponds well with standard tau-phosphohistidine run under the same conditions. Chemical phosphorylation of H4 has been accomplished by using dipotassium phosphoramidate which specifically phosphorylated the imidazole moiety of histidine at neutral pH. NaDodSO4--polyacrylamide gel electrophoresis confirms acid lability, and high-pressure liquid chromatography of protein hydrolysates yields phosphohistidine. 31P NMR of chemically phosphorylated H4 in a structured state reveals two peaks at 4.8 and 7.3 ppm with line widths of 9 and 55 Hz, respectively. These resonances indicate that both histidine residues of H4 (His-18 and His-75) are phosphorylated, the latter relatively immobile and the former relatively free in solution. 31P NMR studies on chemically phosphorylated peptide fragments of H4, namely, H4(1-23) and H4(38-102), confirm this model of H4 structure.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Phosphoric acid solution, NMR reference standard, 85% in D2O (99.9 atom % D), NMR tube size 4.2 mm × 8 in. , WGS-5BL Coaxial NMR tube
Sigma-Aldrich
Phosphoric acid, puriss., ≥99%
Sigma-Aldrich
Phosphoric acid, puriss. p.a., ACS reagent, reag. ISO, reag. Ph. Eur., ≥85%
Sigma-Aldrich
Phosphoric acid, ACS reagent, ≥85 wt. % in H2O
Sigma-Aldrich
Phosphoric acid, ACS reagent, ≥85 wt. % in H2O