Electrical safety during transplantation.

Technologic innovations enable management of medical equipment and power supply systems, with improvements that can affect the technical aspects, economics, and quality of medical service. Herein are outlined some technical guidelines, proposed by Istituto Superiore per la Prevenzione e la Sicurezza del Lavoro, for increasing the effectiveness of the power supply system and the safety of patients and surgeons in the operating room, with particular focus on transplantation. The dependence of diagnoses and therapies on operation of the electrical equipment can potentially cause great risk to patients. Moreover, it is possible that faulty electrical equipment could produce current that may flow through the patient. Because patients are particularly vulnerable when their natural protection is considerably decreased, as during transplantation or other surgery, power supply systems must operate with a high degree of reliability and quality to prevent risk, and must be designed to reduce hazards from direct and indirect contact. Reliability of the power supply system is closely related to the quality of the project, choice of materials, and management of the system (eg, quality and frequency of servicing). Among the proposed guidelines, other than normal referencing, are (1) adoption of a monitoring system to improve the quality of the electrical parameters in the operating room, (2) institution of emergency procedures for management of electrical faults, (3) a procedure for management of fires in the operating room, (4) and maintenance interventions and inspections of medical devices to maintain minimal requirements of safety and performance.

Structural perturbation of the a3-CuB site in mitochondrial cytochrome c oxidase by alcohol solvents.

Ethanol has been observed to cause a perturbation of the catalytic center of the major respiratory protein cytochrome c oxidase. These effects were examined by Fourier transform infrared spectroscopy of carbon monoxide complexes of cytochrome a3Fe and of CuB formed by low-temperature photodissociation of the a3FeCO complex. Carbon monoxide binds to reduced cytochrome oxidase in two major structural forms, alpha and beta, both of which are altered by ethanol. In the absence of ethanol, 15-22% of the total cytochrome oxidase in beef heart mitochondria was observed as beta-forms. Ethanol addition caused a concentration-dependent elimination of the beta-forms with 40% disappearing at 0.05 M (0.23%) ethanol, a concentration that can readily be achieved in the blood of intoxicated individuals. At 0.5 M (2.3%) ethanol and above, almost no beta-forms were detectable. The alpha-CuBCO absorption normally splits into two bands at temperatures below 40 K. This effect was decreased in the presence of ethanol and eliminated by high ethanol concentrations. It appears that ethanol increases the structural fluctuations at the active site of the enzyme, analogous to the effects of increased temperature. There was an 8-10% decrease in the maximum rate of oxygen reduction by mitochondrial cytochrome oxidase in 0.05 M ethanol at 24 degrees C, while higher concentrations of ethanol caused no further inhibition. This is the first demonstration that alpha- and beta-forms of cytochrome c oxidase can be modified by an externally added reagent. Changes in the spectra of alpha-CuBCO in the presence of 50% (v/v) ethylene glycol were quite striking, but variable.(ABSTRACT TRUNCATED AT 250 WORDS)

Energy distributions at the high-spin ferric sites in myoglobin crystals.

The orientation and temperature dependence (4.2-2.5 K) of electron paramagnetic resonance (EPR) power saturation and spin-lattice relaxation rate, and the orientation dependence of signal linewidth, were measured in single crystals of the aquo complex of ferric sperm whale skeletal muscle myoglobin. The spin-packet linewidth was found to be temperature independent and to vary by a factor of seven within the heme plane. An analysis is presented which enables one to arrive at (a) hyperfine component line-widths and, from the in-plane angular variation of the latter, at (b) the widths of distributions in energy differences between low-lying electronic levels and (c) the angular spread in the in-plane principal g-directions. The values of the energy level distributions in crystals obtained from the measurements and analysis reported here are compared with those obtained by a different method for the same protein complex in frozen solution. The spread in the rhombic energy splitting is significantly greater in solution than in the crystal.

Conformational sensitivity of beta-93 cysteine SH to ligation of hemoglobin observed by FT-IR spectroscopy.

The SH vibrational absorption of cysteine F9(beta-93) in concentrated aqueous solutions of native liganded hemoglobin (human HbA, horse, and bovine) has been observed by use of Fourier transform infrared spectroscopy. The pattern of beta-93 SH absorption intensity is ligand dependent. In bovine hemoglobin derivatives the SH absorption intensity pattern is (carbonmonoxy)hemoglobin (HbCO) greater than oxyhemoglobin (HbO2) = cyanomethemoglobin (HbCN) much greater than aquomethemoglobin (metHb) and deoxyhemoglobin (deoxyHb). In horse and human hemoglobin derivatives the pattern is HbCO greater than or equal to HbO2 greater than HbCN greater than metHb. The bovine metHb beta-93 SH shows a much lower absorptivity than that of horse or human metHb, and thus it has a different local tertiary equilibrium conformation than does horse or human hemoglobin. X-ray diffraction studies have shown the beta-93 SH in carbon monoxide or oxygen bound hemoglobin to be situated within a nonpolar pocket between the F, G, and H helices. The higher than usual SH absorption frequency (2592 cm-1) that we observe implies there is no hydrogen bonding for this thiol group while situated within this nonpolar pocket. A similar beta-93 SH absorption has been observed in the beta-chain tetramer (thalassemic hemoglobin H in vivo). The beta-112 SH stretching band, previously observed in the alpha 2 beta 2 tetramer, was observed for the first time in the beta-chain tetramer. A band at 2610 cm-1 that is not due to SH was resolved and found to be ligand dependent.

EPR characterization of alcohol complexes of ferric myoglobin and hemoglobin.

Frozen solution electron paramagnetic resonance spectra of the aquo, methanol, and ethanol complexes of ferric myoglobin and hemoglobin are quantitatively analyzed in terms of the rhombic to tetragonal symmetry ratio and the admixture of quartet states, both with regard to central values of these parameters and the widths of their distributions. In both the methanol and ethanol complexes of ferric myoglobin the main change from the aquo complex is a narrowing of the spread in the rhombic to tetragonal symmetry ratio (reduction in structural variation). The alcohol complexes of both the alpha- and beta-chains within the tetramer of ferric hemoglobin are characterized by a lowering of symmetry (as compared with the aquo complex). Qualitative differences in distribution widths among the complexes are consistent with an origin in molecular structure and dynamics rather than in ice matrix-induced strain.

Alpha and beta forms of cytochrome c oxidase observed in rat heart myocytes by low temperature Fourier transform infrared spectroscopy.

Carbon monoxide bound to myoglobin and cytochrome c oxidase in separated adult rat heart myocytes has been observed with Fourier transform IR spectroscopy at low temperatures. CO complexes of these two proteins can be spectrally separated through temperature manipulation of the relaxation of the photolyzed systems. Photolyzed carboxymyoglobin relaxes very rapidly above 80 K, whereas the CO photolyzed from cytochrome a3 associates with CuB and relaxes very slowly below 140 K. Cytochrome c oxidase is found to be present in two major molecular forms which we designate alpha and beta. Each form contains an a3Fe and its associated CuB which we observe by their CO complexes. The predominant FeCO band, the alpha form of cytochrome oxidase, is similar to that previously seen in beef heart mitochondria, but with a slightly larger activation enthalpy, delta H = 46 kJ/mol. At least one of the beta forms is similar, but two have not been observed in beef heart mitochondria. Upon photolysis of alpha-FeCO, the alpha-CuCO species is formed. This band splits into two at low temperature. Up to half of the FeCO band area of the intact myocytes is distributed among three or more minor species (beta forms). The beta-FeCO bands all appear to be associated with only one beta-CuCO band which does not split at low temperature. After photo-dissociation of CO, the beta forms relax considerably faster than the alpha form, achieving 50% recombination in 10% of the time required for the alpha form. In a tissue slice from an opossum heart exposed to CO, we observed alpha and beta forms of cytochrome oxidase very similar to those in the rat heart myocytes. The cause of the differences between the alpha and beta forms of the enzyme is unknown, but their possible role in the control of respiration is discussed. Carboxymyoglobin contained within intact rat heart myocytes was very similar to sperm whale carboxymyoglobin, but with a much smaller amount of the lower frequency minor component.

Structures of photolyzed carboxymyoglobin.

The structures of photoactivated carboxymyoglobin (Mb*CO) at temperatures to 10 K have been investigated by Fourier transform infrared (FT-IR) spectroscopy, visible spectroscopy, and near-infrared spectroscopy. Two energy states for *CO are observed by FT-IR, which are altered in frequency by 94% and 88% of the difference from the ground-state heme CO toward free CO gas [Alben, J. O., Beece, D., Bowne, S. F., Doster, W., Eisenstein, L. Frauenfelder, H., Good, D., McDonald, J. D., Marden, M. C., Moh, P. P., Reinisch, L., Reynolds, A. H., Shyamsundar, E., & Yue, K. T. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 3744-3748]. Ground-state MbCO shows no absorption in the near-infrared from 700 to 1200 nm. Conversely, Mb*CO shows an absorption near 766 nm, similar to that of ferrous myoglobin (deoxy-Mb) at 758 nm. These data are compared with Mössbauer isomer shifts and quadrupole splitting [Spartalian, K., Lang, G., & Yonetani, T. (1976) Biochim. Biophys. Acta 428, 281-290] and magnetic susceptibility measurements [Roder, H., Berendzen, J., Bowne, S. F., Frauenfelder, H., Sauke, T. B., Shyamsunder, E., & Weissman, M. B. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 2359-2363], which clearly indicate that the iron in both Mb*CO and deoxy-Mb is in the high-spin Fe(II) state, as does the heme transition in the Soret [Iizuka, T., Yamamoto, H., Kotani, M., & Yonetani, T. (1974) Biochim. Biophys. Acta 371, 126-139]. Thus the electronic structure of iron in Mb*CO is nearly identical with that of deoxy-Mb, and *CO is only slightly perturbed from the free gas.(ABSTRACT TRUNCATED AT 250 WORDS)

Dynamic interactions of CO with a3Fe and CuB in cytochrome c oxidase in beef heart mitochondria studied by Fourier transform infrared spectroscopy at low temperatures.

Carbon monoxide bound to cytochrome c oxidase has been observed by Fourier transform infrared spectroscopy between 10 K and 280 K in the dark and during and after continuous photolysis. CO bound to a3Fe absorbs near 1963 cm-1, with minor bands at lower frequencies. Photolysis at low temperatures transfers CO to CuB, with the major component near 2062 cm-1 and a minor one near 2043 cm-1. Vibrational absorptions are assigned by comparison with heme and copper carbonyls, by frequency dependence of all bands on the isotopic mass of CO, and by similar behavior of major and minor components with photolysis and relaxation kinetics as a function of temperature. Reformation of a3FeCO after photolysis is an apparent first order process below 210 K with a distribution of rate constants. The kinetics are well described by a power law. Arrhenius behavior is followed between 140 K and 180 K to yield a peak activation enthalpy of 40.3 kJ/mol and a distribution in g(H) = 2.56 kJ/mol (full width at half-maximum). The major component of a3FeCO shows a very narrow CO absorption band (full width at half-maximum = 2.4 cm-1), while that of CuBCO shows a broader CO absorption (full width at half-maximum = 6 cm-1). These data indicate that in the reduced carbon monoxide complex, a3FeCO is in highly ordered nonpolar surroundings sufficiently separated from CuB that it is not perturbed by motion of the latter, while CuBCO is in less ordered, more flexible surroundings.

Cytochrome oxidase (a3) heme and copper observed by low-temperature Fourier transform infrared spectroscopy of the CO complex.

Carbon monoxide bound to iron or copper in substrate-reduced mitochondrial cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) from beef heart has been used to explore the structural interaction of the a3 heme-copper pocket at 15 K and 80 K in the dark and in the presence of visible light. The vibrational absorptions of CO measured by a Fourier transform infrared interferometer occur in the dark at 1963 cm-1, with small absorptions near 1952 cm-1, and are due to a3 heme--CO complexes. These disappear in strong visible light and are replaced by a major absorption at 2062 cm-1 and a minor one at 2043 cm-1 due to Cu--CO. Relaxation in the dark is rapid and quantitative at 210 K, but becomes negligible below 140 K. The multiple absorptions indicate structural heterogeneity of cytochrome oxidase in mitochondria. The Cu--CO absorptions (vCO) are similar to those in hemocyanin--CO complexes from molluscs (vCO - 2062 cm-1) and crustaceans (vCO = 2043 cm-1). The 2062 cm-1 Cu--CO absorption of cytochrome oxidase is split into two bands at 15 K. Analysis of spectral data suggest the presence of a very nonpolar heme--Cu pocket in which the heme-CO complex is highly ordered, but in which the Cu--CO complex is much more flexible, especially above 80 K. A function for these structures in oxygen reduction is proposed.