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

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Abstract
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% Pi external reference. This peak corresponds well with the chemical shift of standard .pi.-phosphohistidine scanned under similar conditions. Sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis (PAGE) 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 was accomplished by using dipotassium phosphoramidate which specifically phosphorylated the imidazole moiety of histidine at neutral pH. SDS-PAGE confirms acid lability, and high-pressure liquid chromatography of protein hydrolysates yields phosphohistidine. 31P NMR of chemically phosphorylated H4 in a structured state reveals 2 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, i.e., H4(1-23) and H4(38-102), confirm this model of H4 structure.