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

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.

• Authors: J M Fujitaki, G Fung, E Y Oh, R A Smith
• Journal: Biochemistry (Biochemistry) Vol. 20 Issue 12 Pg. 3658-64 (Jun 09 1981) ISSN: 0006-2960 [Print] United States
• PMID: 7196259 (Publication Type: Journal Article, Research Support, U.S. Gov't, Non-P.H.S., Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Histones

Topics

• Animals
• Carcinoma 256, Walker (analysis)
• Cell Nucleus (analysis)
• Histones
• Hydrogen-Ion Concentration
• Kinetics
• Liver (analysis)
• Liver Regeneration
• Magnetic Resonance Spectroscopy
• Phosphorylation
• Protein Conformation