Ring-current shifted proton resonances

The potential of the method of using mutant and chemically modified Hbs to assign proton resonances of Hb is greatly enhanced when correlated with both intensity measurements and calculations from the crystal structure of Hb as determined by X-ray diffraction. A representative example of this approach is El 1 Val in HbCO A. Based on intensity measurements on a mixture of HbCO A and ferrocy-tochrome c, Lindstrom etal. (1972b) have shown for the ring-current-shifted proton resonances at —5.86, -6.48, and -6.58 ppm from HDO that each arises from one CH3 per a(3 dimer. In the spectrum of a mixture of HbCO A and HbCO Sydney [(367(E1 l)Val — Ala],... 

Fig. 13. 250-MHz ring-current-shifted proton resonances of HbCO A and HbCO Sydney (p97Val — Ala). [Adapted from Lindstrom et at. (1972b)].

Fig. 30. Effects of alkyl isocyanides (n-series) on the 600-MHz ring-current-shifted proton resonances of isolated a and p chains of Hb A, Hb A, and sperm whale myoglobin (Mb) in 0.2 M phosphate in D20 at pH 6.6 and 21°C CO, carbon monoxide MNC, methyl isocyanide ENC, ethyl isocyanide nPNC, n-propyl isocyanide nBNC, n-butyl isocyanide. [Adapted from Mims el al. (1983a)].

Resonances in the region — 5 to — 8 ppm from HDO arise from the ring-current-shifted protons due to those protons located above or below the aromatic amino acid residues and the porphyrins of oxy-Hb A (Fig. 8J). (9) Resonances in the region -6 to -20 ppm... 

In the spectrum of ferricytochrome c (Fig. 19) the resonances between —0.5 and —9 ppm correspond to ca. 650 protons of the polypeptide chain. Essentially all the proton resonances of heme c and the axial ligands were found to be shifted by hyperfine interactions with the unpaired electron (Kowalsky (62) Wuthrich (110)), and some ring current-shifted resonances were also outside of the spectral region from DSS to —10 ppm. From a temperature study similar to that shown for MbCN (Figs. 15 and 16) the temperature dependent hyperfine-shifted resonances in Fig. 20 were identified (110). A tentative assignement of some of these lines... 

K. Deoxy-Hb A Ring-Current Shifted and Hyperfine-Shifted Proton Resonances... 

The two CHj groups in isopropyl benzene (Fig. 8) are identical and their resonance is split into a doublet by the adjacent CH, which is deshielded by the ring current. The methine resonance is split into a septet by the six equivalent methyl protons. The aromatic protons are almost equivalent as there is no electronegative group present to affect their chemical shifts. The integral ratios are 5 1 6. 

Representative chemical shifts from the large amount of available data on isothiazoles are included in Table 4. The chemical shifts of the ring hydrogens depend on electron density, ring currents and substituent anisotropies, and substituent effects can usually be predicted, at least qualitatively, by comparison with other aromatic systems. The resonance of H(5) is usually at a lower field than that of H(3) but in some cases this order is reversed. As is discussed later (Section 4.17.3.4) the chemical shift of H(5) is more sensitive to substitution in the 4-position than is that of H(3), and it is also worth noting that the resonance of H(5) is shifted downfield (typically 0.5 p.p.m.) when DMSO is used as solvent, a reflection of the ability of this hydrogen atom to interact with proton acceptors. This matter is discussed again in Section 4.17.3.7. 

In addition to these low field resonances the chemical shift of the coordinated methyl group falls at —0.19 and the methyl portion of the coordinated ethyl group has a chemical shift of —0.61. By comparing these numbers with the chemical shifts of the ethyl protons in the analogous ethyl porphyrin derivative (129), in which the resonance positions are all above —5, it has been concluded that there is little or no ring current in the corrin ligand system. 

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