A novel approach to the study of RBCs based on the collection of three-dimensional high-resolution AFM images and on the measure of the surface roughness of their plasma membrane is presented. The dependence of the roughness from several parameters of the imaging was investigated and a general rule for a trustful analysis and comparison has been suggested. The roughness of RBCs is a morphology-related parameter which has been shown to be characteristic of the single cells composing a sample, but independent of the overall geometric shape (discocyte or spherocyte) of the erythrocytes, thus providing extra-information with respect to a conventional morphology study. The use of the average roughness value as a label of a whole sample was tested on different kinds of samples. Analyzed data revealed that the quantitative roughness value does not change after treatment of RBCs with various commonly used fixation and staining methods while a drastic decrease occurs when studying cells with membrane-skeletal alteration both naturally occurring or artificially induced by chemical treatments. The present method provides a quantitative and powerful tool for a novel approach to the study of erythrocytes structure through an ultrastructural morphological analysis with the potential to give information, in a non-invasive way, on the RBCs function.

A new software procedure, MXAN, to fit experimental XANES spectra is presented here. The method is based on the comparison between the experimental spectrum and several theoretical calculations generated by changing the relevant geometrical parameter of the site around the absorbing atom. The x-ray photoabsorption cross section is calculated using the general multiple-scattering scheme, utilizing a complex Hedin-Lunqvist energy-dependent potential to describe the exchange correlation interaction. Our method has been applied to the study of geometrical environment of the tetrahedral zinc site of the protein superoxide dismutase (SOD). The experimental Zn K-edge XANES spectrum has been fitted in the space of the first shell coordination parameters (ligand distances and angles) following the behavior of the chi-square as a function of the local distortion from the starting crystallographic structure. The recovered structure is found to be independent on the starting conditions, showing the theoretical uniqueness of the structural solution. Strengths and limitations of the application to real systems are also discussed.

The linearly polarized Cu L3-edge x-ray-absorption near-edge structure (XANES) of Bi2Sr2CaCu208+z has been measured and the spectra are interpreted by the full multiple-scattering approach in real space.The polarized spectra over a range of 20 eV can be predicted in terms of the one-electron dipole (Al =+1)transition Cu 2p~ed, probing the unoccupied d-like (1=2) density of states projected on the Cu site with orbital angular momentum m& =0, 1 in the E~~z spectra, and the mI=2, 1, and 0 in the Elc spectra. The oscillator strength for the dipole allowed transitions (Al = -1) Cu 2p~v s is shown to be a factor of 100 weaker than the 2p~3d transitions. The Coulomb interaction in the final state between the Cu 2p core hole and the excited Cu 3d electron is found to be 5.5 eV forming a bound state below the continuum threshold, the well-known Cu L3 white line. On the contrary, the core hole induces a nearly rigid redshift about 1 eV of the high-energy conduction bands.

The Fe(III) --> Fe(II) reduction of the heme iron in aquomet-myoglobin, induced by x-rays at cryogenics temperatures, produces a thermally trapped nonequilibrium state in which a water molecule is still bound to the iron. Water dissociates at T > 160 K, when the protein can relax toward its new equilibrium, deoxy form. Synchrotron radiation x-ray absorption spectroscopy provides information on both the redox state and the Fe-heme structure. Owing to the development of a novel method to analyze the low-energy region of x-ray absorption spectroscopy, we obtain structural pictures of this photo-inducible, irreversible process, with 0.02-0.06-A accuracy, on the protein in solution as well as in crystal. After photo-reduction, the iron-proximal histidine bond is shortened by 0.15 A, a reinforcement that should destabilize the iron in-plane position favoring water dissociation. Moreover, we are able to get the distance of the water molecule even after dissociation from the iron, with a 0.16-A statistical error.

ABSTRACr When the magnetic susceptibility of frozen aqueous solutions of human oxyhemoglobin was measured in the range between 25 and 250 K, it showed a temperatur ependent behavior typical of a thermal equilibrium between a ground singlet state and an excited triplet state for two electrons per heme, the energy separation being 12JI = 146 cm-1. By contrast, within the same temperature range, carboxyhemoglobin was found to be diamagnetic, as already reported.Since the first magnetic studies by Pauling and Coryell (1), the electronic state of iron and oxygen in oxyhemoglobin (HbO2) has been the subject of continuous debate. Five different models for 02 binding in HbO2 have been proposed on the basis of various theoretical interpretations of the available experimental evidence (2)(3)(4)(5)(6).The presence of unpaired electrons in the iron-oxygen bond has been inferred from x-ray fluorescence spectra (7), from spectroscopy data and analogy with cobalt complexes (8, 9), and from M6ssbauer (10) and Raman (11) Optical spectra of the samples were recorded by a Beckman DK-2A spectrophotometer before and after the susceptibility measurement. The concentration of methemoglobin (MetHb) in the samples was estimated to be less than 3% in all cases. To avoid any possible interferences from MetHb, we added KCN to stripped HbO2 to stabilize the MetHb in the low-spin form (15) at any temperature. Measurements were performed on several different preparations of Hb. The temperature-dependent susceptibility was measured at least twice on each sample. RESULTS AND DISCUSSIONTo emphasize the small differences between HbO2 and HbCO samples, all the data were normalized to give the same extrapolation for infinite temperature, which was set as the value extracted from the data of Havemann (16) for HbCO. Typical results for stripped HbO2 and HbCO equilibrated with air, 02 at 1 atm, and CO at 1 atm, are presented in Fig. 1.There is a striking difference in behavior. The stripped HbCO showed a weak Curie law paramagnetic contribution which is quantitatively accounted for by the presence of 0.85% MetHb in the low-spin state.Native HbCO gave similar results, with an even smaller paramagnetic contribution. We conclude that both native and stripped HbCO are diamagnetic. We also conclude that (i) no sample density effects interfered with the volume susceptibility measurements within our resolution and (ii) MetHb, if any, must stay in the same low-spin state in the whole temperature range scanned and strictly obey a simple Curie law.The temperature dependence of the magnetic susceptibility of the frozen solutions of stripped HbO2 was strikingly different from that of HbCO.To fit the data we needed a composite temperature-dependent behavior that allows for the presence of a Curie law paramagnetic component plus a paramagnetic contribution nonlinear in inverse temperature, representing a thermal equilibrium between a ground singlet state and an excited triplet state. The Curie law paramagnetic contribution is easily assigned to the paramagnetis...