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Vicinal proton-proton coupling constants depend on a variety of stereochemical parameters such as bond angle, bond length, substituent electronegativity and orientation, andmost importantlyon the torsion angle between the coupling protons. The sensitivity of vicinal proton-proton coupling constants '.THH, to variations of dihedral angle $ was first rationalized in valence bond terms by Karplus, who showed that the coupling constants could be approximately fitted to a cosz$ function.The Karplus equation 3fHH = Aces*# + Bcos# + C (or variants thereof) has been a major tool of the structural chemist for 35 years. Karplus started with the Ramsey formula for Fermi contact contribution, expressed by ground-state wave functions and a mean excitation energy. More sophisticated molecular orbital (MO) treatments have afforded analogous expressions that may be used to predict trends correctly but are rarely useful for accurate estimations of dihedral angles. Numerous attempts have been made to account for the effect of substituents, bond angles, and lengths -either empirically by the choice of A, B , and C (with different values for each type of compound) or semiempirically, starting with theoretically more sophisticated expressions with statistically adjustable parameters.Theoretical and empirical approaches are reviewed and compared.

The acetylation isoforms of histone H4 from butyratetreated HeLa cells were separated by C 4 reverse-phase high pressure liquid chromatography and by polyacrylamide gel electrophoresis. Histone H4 bands were excised and digested in-gel with the endoprotease trypsin. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry was used to characterize the level of acetylation, and nanoelectrospray tandem mass spectrometric analysis of the acetylated peptides was used to determine the exact sites of acetylation. Although there are 15 acetylation sites possible, only four acetylated peptide sequences were actually observed. The tetra-acetylated form is modified at lysines 5, 8, 12, and 16, the tri-acetylated form is modified at lysines 8, 12, and 16, and the di-acetylated form is modified at lysines 12 and 16. The only significant amount of the mono-acetylated form was found at position 16. These results are consistent with the hypothesis of a "zip" model whereby acetylation of histone H4 proceeds in the direction of from Lys-16 to Lys-5, and deacetylation proceeds in the reverse direction. Histone acetylation and deacetylation are coordinated processes leading to a non-random distribution of isoforms. Our results also revealed that lysine 20 is dimethylated in all modified isoforms, as well as the nonacetylated isoform of H4.

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