Analysis frozen solution spectra

Figure 1. ESR spectra at some temperatures of X-irradiated polycrystalline frozen solutions (c a 1 mol%) of C5H5 and CgH5D, respectively, a) at 15 K. b) simulated spectrum by using the parameters shown in Table 4. c) 05 150 " " at 30 K in CFCI3. d) simulation using hyperfme coupling data shown in Table 4 for a mixture of component 1 component 2 of C6H5D . The ratio 1 2 = 0.38 0.62 obtained from the component analysis of Figure 2 was employed.

Thereafter, crystals were brought back to the aerobic 25% MPD solution, buffered with 50 mAf sodium phosphate, pH 5.5. This procedure is based on Avigliano et al. s (157) method of preparing T2D ascorbate oxidase in solution and was modified by Merli et al. (159) for use with ascorbate oxidase crystals. The 2.5-A-resolution X-ray structure analysis by difference-Fourier techniques and crystallographic refinement shows that about 1.3 copper ions per ascorbate oxidase monomer are removed. The copper is lost from all three copper sites of the trinuclear copper species, whereby the EPR-active type-2 copper is the most depleted (see Fig. 10). Type-1 copper is not affected. The EPR spectra from polycrystalline samples of the respective native and T2D ascorbate oxidase were recorded. The native spectrum exhibits the type-1 and type-2 EPR signals in a ratio of about 1 1, as expected from the crystal structure. The T2D spectrum reveals the characteristic resonances of the type-1 copper center, also observed for T2D ascorbate oxidase in frozen solution, and the complete disappearance of the spectroscopic type-2 copper. This observation indicates preferential formation of a Cu-depleted form with the holes equally distributed over all three copper sites. Each of these Cu-depleted species may represent an anti-ferromagnetically coupled copper pair that is EPR-silent and that could explain the disappearance of the type-2 EPR signal. 

Double integration of the area under the first derivative reveals that 48 =i= 2% of the chemically determined copper (IS) is EPR detectable in frozen solution. Table I summarizes the experimental g and A values measured from the recorded spectra. A best fit of the EPR spectrum of oxidized ascorbate oxidase is obtained by computer simulation, using the high-frequency measurements at 35 GHz (28). The ratio of type 1 to type 2 copper is estimated by double integration of the first low-field line, which arises from the type 2 copper, at approximately 0.270 T (IS). Roughly 25% of the EPR-detectable copper in ascorbate oxidase is type 2, whereas 75% is blue type 1 copper. This ratio is confirmed by computer analysis (IS) and agrees with earlier results (28) (Figure 4). 

In chemical shift calculations for acylium ions, it was not necessary to model the ionic lattice to obtain accurate values. These ions have tetravalent carbons with no formally empty orbitals, as verified by natural bond orbital calculations (89). Shift calculations for simple carbenium ions with formally empty orbitals may require treatment of the medium. We prepared the isopropyl cation by the adsorption of 2-bromopropane-2-13C onto frozen SbF5 at 223 K and obtained a 13C CP/MAS spectrum at 83 K (53). Analysis of the spinning sidebands yielded experimental values of = 497 ppm, 822 = 385 ppm, and (%3 = 77 ppm. The isotropic 13C shift, 320 ppm, is within 1 ppm of the value in magic acid solution (17). Other NMR evidence includes dipolar dephasing experiments and observation at higher temperature of a scalar doublet ( c-h = 165 Hz) for the cation center. 

N- )onor Ligands. The triplet-state X-band e.s.r. spectrum of the oxo-vanadium(iv)-chelate of tetrakis(aminomethyl)methane has been recorded for frozen aqueous-glycol solutions at 77 K. The spiro conformation of the methylene groups of this ligand ensure that the two metal centres in this dinuclear complex will have a non-parallel alignment of their -tensor axes. An analysis... 

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