Parts I and II (Perutz et al. (1974), Biochemistry 13, 2163, 2174) presented evidence indicating that inositol hexaphosphate (IHP) is capable of switching the quaternary structure of certain high-spin ferrous and ferric hemoglobins from the oxy (R) to the deoxy (T) state. Here we analyze the resulting changes in visible and near-infrared absorption spectra, paramagnetic susceptibility, and paramagnetically shifted proton and electron spin resonances. In aquo-and hydroxymethemoglobin where components of high and low spin coexist in thermal equilibrium, IHP causes changes in the absorption spectra and paramagnetically shifted proton resonances indicative of a shift of the equilibrium to higher spin. This is confirmed by a stoichiometric rise in the paramagnetic susceptibility of aquomethemoglobin solutions by up to 6.4 % at concentrations of 1 mol of IHP/mol of tetramer. Other spectral changes produced by IHP include a rise in intensity of the Soret band and red shifts of all high-spin bands. The spectral changes are identical with those observed by Perutz ((1972), Nature (London) 237, 495) on dissociation of carbon monoxide from valency hybrids. In fluoromethemoglobin which is probably pure high spin, IHP causes red shifts of all bands, a rise in intensity of the Soret band and a fall in all others. In iron-porphyrin complexes which are in thermal equilibrium between two different spin states, a shift toward F 1 evidence presented in parts I and II (Perutz et al., 1974a,b) indicated that inositol hexaphosphate (IHP)1 is capable of converting high-spin hemoglobin derivatives from the quaternary R to the T structure, and that this transition is accompanied by marked spectral changes. IHP was also found to cause spectral changes in several low-spin derivatives, but these were weaker and did not appear to be associated with transitions between the two quaternary structures. In this paper we examine in detail the changes in electronic heme spectra, paramagnetic susceptibility, paramagnetically shifted proton resonances, and electron spin resonances produced in methemoglobin by IHP.The derivatives we have examined include fluoromethemot From the
Solutions of DNA, spin-labelled with the radical cation of chlorpromazine, were used to produce oriented species of fibres pulled from a gel obtained by ultracentrifugation. The electron spin resonance spectra, recorded at X and Q band frequencies, are given for both gel and fibres; 14N hyperfine coupling parameters were obtained by computer fitting. The spectra are explained in terms of a strongly immobilized label having one principal hyperfine tensor axis parallel to the axis of the DNA helix, and the preferential orientation of the chlorpromazine ions with their planes perpendicular to the DNA helical axis. Ohnishi and McConnell [l] have suggested that the psychotropic activity of the tranquilizer chlorpromazine could be connected with the binding of the radical cation chlorpromazine onto DNA. In paticular they have presented evidence from electron spin resonance (ESR) studies of DNA/chlorpromazine ion complexes which is consistent with the possibility of the chlorpromazine ion intercalating between the base pairs of the DNA.The question of intercalation as applied to this nonaromatic species is of particular interest because the chlorpromazine molecule and ion are not quite planar, characteristic of many sulphur complexes of this type [2,3], and the current ideas on intercalation do not make explicit provision for such special cases [4,5].In order to investigate this problem further a series of measurements have been made on fibres of DNA/ chlorpromazine ion complexes; as all the previous studies had been carried out on solutions, orientational information was available only in a restricted form from flow experiments. In this study it has been possible to obtain the detailed angular variation of the ESR spectrum with respect to the fibre axis. The spectra are presented from both X and Q band (9.5 GHz and 32.8 GHz) measurements. In addition, the study of the fibres permits direct comparison with other techniques and allows the system to be studied under a variety of conditions which are not possible with solution studies.The ESR results, coupled with a theoretical synthesis, are consistent with a preferred orientation of the chlorpromazine ion perpendicular to the fibre axis, AhhrPriution. ESR, electron spin resonance. Trivicd Name. Chlorpromazine, 2-chloro-l0-(3-dimethylaminopropy1)-phenothiazine.but it is also apparent that valuable information might be obscured by a large degree of misorientation which exists in both fibresand in the previous flow experiments. EXPERIMENTAL PROCEDUREThe complexes were made from high-molecularweight calf-thymus DNA (Sigma type V average molecular weight 1 to 1.3 million) and chlorpromazine hydrochloride. (May and Baker)The chlorpromazine was oxidised with equimolar quantities of sodium persulphate and added to the aqueous solution of DNA, to produce phosphate : drug ratios of about 6.0. The DNA concentration was about 0.7 mM (total base concentration) and the ionic strength 0.04 M (NaCI). In order to increase the stability of the radical cation the solution was not buffere...
Details of the way in which haemplane orientations were deduced from g -value measurements on five different crystal types of acid-met myoglobin were given in parts I and I I (Bennett, Gibson & Ingram 1957, and Bennett et al . 1961). This paper now summarizes more detailed measurements of line-width and g -value variations observed in both the acid-met and azide derivatives of type A myoglobin crystals. Since these crystals are those for which a detailed X-ray analysis is now available, a direct comparison of this with the electron resonance measurements can now be made. The g -values obtained for the azide derivative are first analysed, and their anisotropy and asymmetry are related to the possible orientation of the azide group itself. The line widths of the electron resonance absorptions, and their angular variations are then summarized and discussed. It is shown that their large magnitudes and rapid variation with angle can be explained in terms of a slight random misorientation of the molecular axes within the crystal, and that, due to the large g -value anisotropy in the acid-met derivative, a standard deviation of only 1.6° in angular distribution is sufficient to explain the results obtained. A similar analysis is also applied to the results on the azide derivative.
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