Hypochlorous acid-induced oxidative damage of human red blood cells: effects of tert-butyl hydroperoxide and nitrite on the HOCl reaction with erythrocytes

Hypochlorous acid, one of the most powerful biological oxidants, is believed to be important in the pathogenesis of some diseases. The purpose of this study was to further characterise the membrane and intracellular events which resulted in HOCl-induced oxidative impairments and haemolysis of human erythrocytes and interaction of different oxidative agents, which accumulated during respiratory burst, in the process of RBS oxidation. The sequence of cellular events after red blood cell exposure to HOCl: cell morphological transformations, oxidation of cellular constituents, enzyme modifications, and haemolysis have been evaluated. It was shown that HOCl-treated cells underwent colloid-osmotic haemolysis, preceded by rapid morphological transformations and membrane structural transitions. The activation energy of the process of haemolysis (after removal of the excess of oxidative agent) was estimated to be 146+/-22 kJ/mol at temperatures above the break point of Arrhenius plot (31-32 degrees C). This value corresponds to the activation energy of the process of protein denaturation. Modification of erythrocytes by HOCl inhibited membrane acetylcholinesterase (uncompetitive type of inhibition), depleted intracellular glutathione, activated intracellular glutathione peroxidase, but did not induce membrane lipid peroxidation. The presence of other oxidants, nitrite or tert-butyl hydroperoxide (t-BHP), promoted the oxidative damage induced by HOCl and led to new oxidative reactions.     

Hypochlorous acid-induced membrane pore formation in red blood cells

The hyperproduction of hypochlorous acid (HOCl), an extremely toxic biological oxidant generated by neutrophils and monocytes, is involved in the pathogenesis of many diseases. In these studies, we attempted to determine the membrane and cellular events associated with HOCl-induced erythrocyte impairment and haemolysis. In vitro human erythrocyte exposure to HOCl (0.1-1.0 mM) resulted in rapid oxidation of reduced glutathione, an increase in cell osmotic fragility and the formation of transient membrane pores. The process of glutathione oxidation depended on the [oxidant]/[cell number] ratio. The HOCl-induced haemolysis observed was apparently mediated by pore formation and altered membrane electrolyte permeability. The estimated pore radius was approximately 0.7 nm and the average number per cell was 0.01. The rate constant of HOCl-produced haemolysis depended on pH. There were significant differences in haemolysis of HOCl-treated erythrocytes which had maximal stability at pH 7.2-7.3.     

The mechanism of Zn-phthalocyanine photosensitized lysis of human erythrocytes

The phthalocyanines have recently been suggested as one of most effective possible sensitizers for photodynamic therapy and the blood viral inactivation. The further characterisation of the mechanism of human red blood cell lysis and membrane alterations upon photodynamic treatment in the presence of Zn-phthalocyanine was the aim of this study. It was found that there were (2.7+/-0.4).10(7) dye binding sites per red blood cell with the association constant equal to (1.4+/-0.3).10(4) M(-1). Two types of the photosensitized haemolysis: haemolysis during irradiation ("light" haemolysis) and post-irradiation haemolysis ("dark" haemolysis) were studied. The erythrocyte membrane hyperpolarisation, membrane fluidisation and cell swelling preceded the "light" haemolysis. The modification of the erythrocyte membrane band 3 protein by DIDS (an inhibitor of anion exchange) increased the rate of the "light" haemolysis. The rate of "dark" haemolysis was higher and that of "light" haemolysis was lower in potassium media in comparison to sodium ones. The rates of photohaemolysis depended on the erythrocyte membrane potential: a decrease of membrane potential inhibited both types of haemolysis. The cell shrinkage in the presence of sucrose (up to 15 mM) inhibited the "dark" haemolysis but significantly increased the "light" haemolysis. Oxidation of intracellular oxyHb to metHb by nitrite, which drastically decreases intracellular oxygen concentration, as well as GSH concentration, inhibited the rate of the "light" haemolysis. The results allow for the conclusion that the mechanism of photochemical ("light") haemolysis is not of a colloid-osmotical type, in contrast to the post-irradiation ("dark") haemolysis. The photochemical oxidation or denaturation of band 3 protein plays a significant role in the formation of haemolytic holes. The membrane lipid peroxidation, as well as glutathione oxidation, does not participate in the process of photosensitized haemolysis. From the inhibition of "dark" haemolysis by sucrose the apparent pore radius was estimated to be about 1.1 nm. The pores appear to be transient short-lived ones, the average pore number per cell was 0.02.     

Detection of erythrocyte membrane structural abnormalities in lecithin: cholesterol acyltransferase deficiency using a spin label approach.

The membrane fluidity of erythrocytes from patients with Lecithin: cholesterol acyltransferase (LCAT) deficiency was studied by means of electron spin resonance. The temperature dependence of the separation of the outer extrema of the spectra of 2-(3-carboxy-propyl)-4,4-dimethyl, 2-tridecyl-3-oxazolidinyloxyl spin probe was monitored for normal, presumed carrier and clinically affected subjects. The temperature profile of controls was significantly different from that of the presumed carriers and the clinically affected individuals. The results show that the compositional abnormalities previously noted in erythrocyte membranes from patients with LCAT deficiency are associated with alterations in the physiocochemical state of the membrane. An investigation of the spectral lineshapes below 10 degrees C allowed a distinction to be made at the membrane level between clinically affected subjects and clinically normal heterozygous carriers. Alterations in the temperature dependence of elec-ron spin resonance parameters may provide a sensitive index of red cell membrane alterations in pathological states of generalized membrane involvement.     

Effect of UVA radiation on membrane fluidity and radical decay in human fibroblasts as detected by spin labeled stearic acids

The ultraviolet A (UVA) radiation component of sunlight (320-400 nm) has been shown to be a source of oxidative stress to cells via generation of reactive oxygen species. We report here some consequences of the UVA irradiation on cell membranes detected by electron paramagnetic resonance (EPR) spectroscopy. Paramagnetic nitroxide derivatives of stearic acid bearing the monitoring group at different depths in the hydrocarbon chain were incorporated into human fibroblasts membranes to analyze two main characteristics: kinetics of the nitroxide reduction and membrane fluidity. These two characteristics were compared for control and UVA-irradiated (0-250 kJ/m(2)) cells. The term relative redox capacity (RRC) was introduced to characterize and to compare free radical reduction measured by EPR with some well-known viability/clonogenicity tests. Our results showed that UVA-irradiation produces a more rigid membrane structure, especially at higher doses. Furthermore, we found that trends agree in survival measured by neutral red (NR), trypan blue (TB), and clonogenic efficiency compared with RRC values measured by EPR for low and medium exposure doses. Above 100 kJ/m(2), differences between these tests were observed. Antioxidant effect was modeled by alpha-tocopherol-acetate treatment of the cells before UVA irradiation. While NR, TB and clonogenicity tests showed protection at the highest UVA doses (>100 kJ/m(2)), results obtained with EPR measurements, both membrane fluidity and kinetics, or using MTT test did not exhibit this protective effect.     

Effect of temperature on surface properties of cervical tissue homogenate and organic phase monolayers

The temperature dependence of Langmuir monolayers of normal and cancerous human cervical tissues and their organic phases between temperatures of 37 and 45 degrees C was evaluated. Analysis of the surface pressure-area isotherms revealed significantly different increase in fluidity of the cancerous cervical tissue monolayer at 42 degrees C as opposed to the normal cervical tissue monolayers (p<0.05). Similarly, in the case of cervical cancerous organic phase monolayers significant increase of fluidity was observed at 40 degrees C whereas no such change was observed in the normal cervical organic phase monolayers. The effect of temperature was found to be different in cancerous and normal cervical tissues and this may be due to the different lipid profiles in them. Cancerous cervical tissues had 1.8-fold higher total lipids as compared to the normals. Similarly, the PC, PE, PI, PG, SM and PS levels in cancerous cervical tissues were 3.6, 2.0, 2.3, 4.7, 1.7 and 2.2 times higher than those of normal cervical tissues, respectively. Significant cancer-normal difference in minimum surface tension and hysteresis area was found at all temperatures studied for both tissue homogenates and organic phases. For example, cancerous tissue homogenates showed minimum surface tensions of 51.9+/-4.6, 54.4+/-5.9, 57.6+/-6.0 and 51.9+/-5.6mN/m at temperatures 37, 40, 42 and 45 degrees C whereas the corresponding values for normal cervical tissue homogenates were 39.3+/-3.6, 39.2+/-3.7, 39.2+/-3.8 and 39.1+/-3.6, respectively. The fluidity change at hyperthermic range of temperature can be correlated to the increased efficiency of drug on combination therapy with hyperthermia. These results may have implications in manipulating the fluidity of cervical cancer tissue membranes for better permeability thereby leading to better therapeutic strategies for cervical cancer.     

The temperature effect during pulse application on cell membrane fluidity and permeabilization

Cell membrane permeabilization is caused by the application of high intensity electric pulses of short duration. The extent of cell membrane permeabilization depends on electric pulse parameters, characteristics of the electropermeabilization media and properties of cells exposed to electric pulses. In the present study, the temperature effect during pulse application on cell membrane fluidity and permeabilization was determined in two different cell lines: V-79 and B16F-1. While cell membrane fluidity was determined by electron paramagnetic resonance (EPR) method, the cell membrane electropermeabilization was determined by uptake of bleomycin and clonogenic assay. A train of eight rectangular pulses with the amplitude of 500 V/cm, 700 V/cm and 900 V/cm in the duration of 100 micros and with repetition frequency 1 Hz was applied. Immediately after the pulse application, 50 microl droplet of cell suspension was maintained at room temperature in order to allow cell membrane resealing. The cells were then plated for clonogenic assay. The main finding of this study is that the chilling of cell suspension from physiological temperature (of 37 degrees C) to 4 degrees C has significant effect on cell membrane electropermeabilization, leading to lower percent of cell membrane permeabilization. The differences are most pronounced when cells are exposed to electric pulse amplitude of 900 V/cm. At the same time with the decreasing of temperature, the cell membranes become less fluid, with higher order parameters in all three types of domains and higher proportion of domain with highest order parameter. Our results indicate that cell membrane fluidity and domain structure influence the electropermeabilization of cells, however it seems that some other factors may have contributing role.     

Energetics of cresols and of methylphenoxyl radicals

Combustion calorimetry studies were used to determine the standard molar enthalpies of formation of o-, m-, and p-cresols, at 298.15 K, in the condensed state as Delta(f)H(m) degrees (o-CH(3)C(6)H(4)OH,cr) = -204.2 +/- 2.7 kJ.mol(-1), Delta(f)H(m) degrees (m-CH(3)C(6)H(4)OH,l) = -196.6 +/- 2.1 kJ.mol(-1), and Delta(f)H(m) degrees (p-CH(3)C(6)H(4)OH,cr) = -202.2 +/- 3.0 kJ.mol(-1). Calvet drop calorimetric measurements led to the following enthalpy of sublimation and vaporization values at 298.15 K: Delta(sub)H(m) degrees (o-CH(3)C(6)H(4)OH) = 73.74 +/- 0.46 kJ.mol(-1), Delta(vap)H(m) degrees (m-CH(3)C(6)H(4)OH) = 64.96 +/- 0.69 kJ.mol(-1), and Delta(sub)H(m) degrees (p-CH(3)C(6)H(4)OH) = 73.13 +/- 0.56 kJ.mol(-1). From the obtained Delta(f)H(m) degrees (l/cr) and Delta(vap)H(m) degrees /Delta(sub)H(m) degrees values, it was possible to derive Delta(f)H(m) degrees (o-CH(3)C(6)H(4)OH,g) = -130.5 +/- 2.7 kJ.mol(-1), Delta(f)H(m) degrees (m-CH(3)C(6)H(4)OH,g) = -131.6 +/- 2.2 kJ.mol(-1), and Delta(f)H(m) degrees (p-CH(3)C(6)H(4)OH,g) = -129.1 +/- 3.1 kJ.mol(-1). These values, together with the enthalpies of isodesmic and isogyric gas-phase reactions predicted by the B3LYP/cc-pVDZ, B3LYP/cc-pVTZ, B3P86/cc-pVDZ, B3P86/cc-pVTZ, MPW1PW91/cc-pVTZ, CBS-QB3, and CCSD/cc-pVDZ//B3LYP/cc-pVTZ methods, were used to obtain the differences between the enthalpy of formation of the phenoxyl radical and the enthalpies of formation of the three methylphenoxyl radicals: Delta(f)H(m) degrees (C(6)H(5)O*,g) - Delta(f)H(m) degrees (o-CH(3)C(6)H(4)O*,g) = 42.2 +/- 2.8 kJ.mol(-1), Delta(f)H(m) degrees (C(6)H(5)O*,g) - Delta(f)H(m) degrees (m-CH(3)C(6)H(4)O*,g) = 36.1 +/- 2.4 kJ.mol(-1), and Delta(f)H(m) degrees (C(6)H(5)O*,g) - Delta(f)H(m) degrees (p-CH(3)C(6)H(4)O*,g) = 38.6 +/- 3.2 kJ.mol(-1). The corresponding differences in O-H bond dissociation enthalpies were also derived as DH degrees (C(6)H(5)O-H) - DH degrees (o-CH(3)C(6)H(4)O-H) = 8.1 +/- 4.0 kJ.mol(-1), DH degrees (C(6)H(5)O-H) - DH degrees (m-CH(3)C(6)H(4)O-H) = 0.9 +/- 3.4 kJ.mol(-1), and DH degrees (C(6)H(5)O-H) - DH degrees (p-CH(3)C(6)H(4)O-H) = 5.9 +/- 4.5 kJ.mol(-1). Based on the differences in Gibbs energies of formation obtained from the enthalpic data mentioned above and from published or calculated entropy values, it is concluded that the relative stability of the cresols varies according to p-cresol < m-cresol < o-cresol, and that of the radicals follows the trend m-methylphenoxyl < p-methylphenoxyl < o-methylphenoxyl. It is also found that these tendencies are enthalpically controlled.     

Effect of phenylhydrazine-induced structural alterations of human erythrocytes on basic drug penetration

The phospholipid organization, lipid fluidity and spectrin degradation were measured in human erythrocytes oxidized with phenylhydrazine, and the contribution of these structural alterations to the penetration of perazine and promethazine into the membrane was estimated. It was found that exposure of erythrocytes to phenylhydrazine (0.2--0.4 mg/ml) produced a 35--40% decrease in the amount of spectrin, with resultant gross morphological changes to the echinocyte conformation. The phosphatidylethanolamine content in the treated erythrocytes was greatly lowered compared with that in the untreated cells. Treatment with phenylhydrazine (0.05--0.2 mg/ml) dramatically diminished the lipid fluidity of the membrane, as estimated by electron spin resonance (ESR) spectrometry, and the ESR study revealed increased restriction of the molecular motion of the hydrophobic core in the treated membrane. These results suggest a drastic alteration of the erythrocyte membrane structure. The amount of drugs which penetrated into the treated erythrocytes increased markedly with increasing phenylhydrazine concentration, suggesting enhanced membrane permeability and facilitated localization of the drugs in the hydrophobic regions due to the structural changes and partial disturbance of the lipid organization.     

The thermodynamics of the transformation of graphite to multiwalled carbon nanotubes

The thermodynamic quantities associated to the transformation from graphite to multiwalled carbon nanotubes (MWCNTs) were determined by electromotive force (emf) and differential scanning calorimetry (DSC) measurements. From the emf versus T data of galvanic cell Mo|Cr(3)C(2), CrF2, MWCNTs|CaF2 s.c.|Cr(3)C(2), CrF2, graphite|Mo with CaF2 as solid electrolyte, Delta(r)H(T) degrees= 8.25 +/- 0.09 kJ mol(-1) and Delta(r)S(T) degrees= 11.72 +/- 0.09 JK(-1) mol(-1) were found at average temperature T = 874 K. The transformation enthalpy was also measured by DSC of the Mn(7)C(3) formation starting from graphite or MWCNTs. Thermodynamic values at 298 K were calculated to be: Delta(r)H(298) degrees = 9.0 +/- 0.8 kJ mol(-1) as averaged value from both techniques and Delta(r)S(298) degrees approximately Delta(r)S(T) degrees. At absolute zero, the residual entropy of MWCNTs was estimated 11.63 +/- 0.09 JK(-1) mol(-1), and transformation enthalpy Delta(r)H(0) degrees approximately Delta(r)H(298) degrees. The latter agrees satisfactorily with the theoretical calculations for the graphite-MWCNTs transformation. On thermodynamic basis, the transformation becomes spontaneous above 704 +/- 13 K.     

Phonons and thermodynamics of unmixed and disordered Li0.6FePO4

The lithium-storage material Li(0.6)FePO(4) was studied by inelastic neutron scattering and differential scanning calorimetry. Li(0.6)FePO(4) undergoes a transformation from a two-phase mixture (heterosite and triphylite) to a disordered solid-solution at 200 degrees C. Phonon densities of states (DOS) obtained from the inelastic neutron scattering were similar for the two-phase sample measured at 180 degrees C and the disordered sample measured at 220 degrees C. The vibrational entropy of transformation is 1.8 +/-0.9 J/(K mol), which is smaller than the configurational entropy difference of approximately 3.1 J/(K mol). The measured enthalpy of the disordering transition was estimated as 2.5 kJ/mol. The phonon data show a small change in lattice dynamics upon disordering.     

Hydroxylation of phenolic compounds by a peroxodicopper(II) complex: further insight into the mechanism of tyrosinase

The dicopper(I) complex [Cu2(MeL66)]2+ (where MeL66 is the hexadentate ligand 3,5-bis-{bis-[2-(1-methyl-1H-benzimidazol-2-yl)-ethyl]-amino}-meth ylbenzene) reacts reversibly with dioxygen at low temperature to form a mu-peroxo adduct. Kinetic studies of O2 binding carried out in acetone in the temperature range from -80 to -55 degrees C yielded the activation parameters DeltaH1(not equal) = 40.4 +/- 2.2 kJ mol(-1), DeltaS1)(not equal) = -41.4 +/- 10.8 J K(-1) mol(-1) and DeltaH(-1)(not equal) = 72.5 +/- 2.4 kJ mol(-1), DeltaS(-1)(not equal) = 46.7 +/- 11.1 J K(-1) mol(-1) for the forward and reverse reaction, respectively, and the binding parameters of O2 DeltaH degrees = -32.2 +/- 2.2 kJ mol(-1) and DeltaS degrees = -88.1 +/- 10.7 J K(-1) mol(-1). The hydroxylation of a series of p-substituted phenolate salts by [Cu2(MeL66)O2]2+ studied in acetone at -55 degrees C indicates that the reaction occurs with an electrophilic aromatic substitution mechanism, with a Hammett constant rho = -1.84. The temperature dependence of the phenol hydroxylation was studied between -84 and -70 degrees C for a range of sodium p-cyanophenolate concentrations. The rate plots were hyperbolic and enabled to derive the activation parameters for the monophenolase reaction DeltaH(not equal)ox = 29.1 +/- 3.0 kJ mol(-1), DeltaS(not equal)ox = -115 +/- 15 J K(-1) mol(-1), and the binding parameters of the phenolate to the mu-peroxo species DeltaH degrees(b) = -8.1 +/- 1.2 kJ mol(-1) and DeltaS degrees(b) = -8.9 +/- 6.2 J K(-1) mol(-1). Thus, the complete set of kinetic and thermodynamic parameters for the two separate steps of O2 binding and phenol hydroxylation have been obtained for [Cu2(MeL66)]2+.     

Kinetics and thermodynamics of the reaction of deoxyhemerythrin with nitric oxide

Deoxyhemerythrin reacts with NO to form a 1:1 adduct shown spectrophotometrically. The kinetics of the formation have been studied directly by stopped-flow measurements at four different temperatures (0.0-23.6 degrees C). The kinetics of the dissociation have been studied, also by stopped-flow techniques, at five different temperatures (4.0-35.1 degrees C) using three different scavengers [Fe(II)(edta)2-, O2 and sperm whale deoxymyoglobin], which gave similar values. For the formation kf = (4.2 +/- 0.2) x 10(6) M-1 s-1, delta Hf not equal = 44.3 +/- 2.3 kJ mol-1, delta Sf not equal to = 30 +/- 8 J mol-1 K-1 and for the dissociation kd = 0.84 +/- 0.02 s-1, delta Hd not equal to 95.6 +/- 2.1 kJ mol-1 delta Sd not equal to = 74 +/- 7 J mol-1 K-1 (25 degrees C, I = 0.2 M and pH 7-8.1). From the kinetic data the thermodynamic data for the formation of HrNO were calculated: Kf = (5.0 +/- 0.3) x 10(6) M-1, delta H = -51.3 +/- 3.1 kJ mol-1 and delta S = -44 +/- 11 J mol-1 K-1 (25 degrees C). The kinetic data suggest that NO occupies the same iron(II) site in deoxyhemerythrin as oxygen does. The equilibrium constant for the formation of Fe(II)(edta)(NO)2- has been redetermined: K1 = (1.45 +/- 0.07) x 10(7) M-1, delta H = -77.5 +/- 1.5 kJ and mol-1 and delta S = -123.5 J mol-1 K-1 (25 degrees C).     

Gas-phase protonation thermochemistry of arginine

The gas-phase basicity (GB), proton affinity (PA), and protonation entropy (DeltapS degrees (M)=S degrees (MH+)-S degrees (M)) of arginine (Arg) have been experimentally determined by the extended kinetic method using an electrospray ionization quadrupole time-of-flight (ESI-Q-TOF) mass spectrometer. This method provides GB(Arg)=1004.3+/-2.2 (4.9) kJ.mol(-1) (indicated errors are standard deviations, and in parentheses, 95% confidence limits are given). Consideration of previous experimental data using a fast atom bombardment ionization tandem sector mass spectrometer slightly modifies these estimates since GB(Arg)=1005.9+/-3.1 (6.6) kJ.mol(-1). Lower limits of the proton affinity, PA(Arg)=1046+/-4 (7) kJ.mol(-1), and of the "protonation entropy", DeltapS degrees (Arg)=S degrees (ArgH+)-S degrees (Arg)=-27+/-7 (15) J.mol(-1).K(-1), are also provided by the experiments. Theoretical calculations conducted at the B3LYP/6-311+G(3df,2p)//B3LYP/6-31+G(d,p) level, including 298 K enthalpy correction, predict a proton affinity value of ca. 1053 kJ.mol-1 after consideration of isodesmic proton-transfer reactions with guanidine as the reference base. Computations including explicit treatment of hindered rotations and mixing of conformers confirm that a noticeable entropy loss does occur upon protonation, which leads to a theoretical DeltapS degrees (Arg) term of ca. -45 J.mol(-1).K(-1). The following evaluated thermochemical parameter values are proposed: GB(Arg)=1005+/-3 kJ.mol(-1); PA(Arg)=1051+/-5 kJ.mol(-1), and DeltapS degrees (Arg)=-45+/-12 J.mol(-1).K(-1).     

Gas-phase basicity of methionine

Proton affinity and protonation entropy of methionine (Met) were determined by the extended kinetic method from ESI-Q-TOF tandem mass spectrometry experiments. The values, PA(Met) = 937.5 +/- 2.9 kJ mol(-1) and Delta(p)S degrees (Met) = - 22 +/- 5 J mol(-1) K(-1), lead to gas-phase basicity GB(Met) = 898.2 +/- 3.2 kJ.mol(-1). Quantum chemical calculations using density functional theory confirm that the proton affinity of Met is indeed in the 940 kJ mol(-1) range and that a significant entropy loss, of at least - 25 J mol(-1) K(-1), occurs upon protonation. This last point is evidenced here for the first time and suggests revision of the tabulated protonation thermochemistry of Met. A comparison with previous experimental data allows us to propose the following evaluated thermochemical values: PA(Met) = 943 +/- 4 kJ mol(-1) and Delta(p)S degrees (Met) = - 35 +/- 15 J mol(-1) K(-1) and GB(Met) = 900 +/- 2 kJ mol(-1).     

Rate constant and activation energy measurement for the reaction of atomic hydrogen with thiocyanate and azide in aqueous solution

Arrhenius parameters for the reaction of hydrogen atoms with azide and thiocyanate in aqueous solution have been determined using electron pulse radiolysis and electron paramagnetic resonance free induction decay attenuation measurements. Absolute values for SCN-, N3(-), and HN3 were well-described over the temperature range of 9-81 degrees C by the equations log k5 = (12.03 +/- 0.12) - [(21.05 +/- 0.66 kJ mol(-1))/2.303RT], log k10 = (12.75 +/- 0.21) - [(18.43 +/- 1.22 kJ mol(-1))/2.303RT], and log k15 = (11.59 +/- 0.12) - [(21.44 +/- 0.69 kJ mol(-1))/2.303RT], corresponding to room temperature (22 degrees C) rate constants of (2.07 +/- 0.03) x 10(8), (3.15 +/- 0.08) x 10(9), and (6.31 +/- 0.05) x 10(7) M(-1) s(-1) and activation energies for these chemicals of 21.05 +/- 0.66, 18.4 +/- 1.2, and 21.44 +/- 0.69 kJ mol(-1), respectively. The similarity of these three measured activation energies, taken together with the available information on reaction products, suggests a similar reaction mechanism, which is proposed to be an initial hydrogen atom adduct formation in these molecules, followed by single bond breakage.     

Characterization of two types of mitochondrial creatine kinase isolated from normal human cardiac muscle and brain tissue

Two types of mitochondrial creatine kinase (Mi-CK), sarcomeric (sMi-) and ubiquitous (uMi-)CKs, were isolated from normal human cardiac muscle and brain tissue, respectively, and their heterogeneity was characterized by means of isoelectric focusing (IEF). Octameric sMi-CK and uMi-CK were electrophoresed cathodic to cytoplasmic muscle-type creatine kinase isoenzyme (CK-MM) and dimeric Mi-CKs were found at the position of CK-MM on a cellulose acetate membrane. The electrophoretic mobilities of sMi-CK were similar to those of uMi-CK. Octameric sMi-CK was focused at pI 7.1-8.0 and dimeric forms at pI 6.55, 6.75, 6.85, and 6.95. New bands appearing at pI 6.65 and 6.75 after treatment of sMi-CK with carboxypeptidase B were found to be delysined forms. sMi-CK reacted with anti-sMi-CK antibodies, and the immune complexes were focused at pI 5.8. The Km value of sMi-CK for creatine phosphate (PCr) was 1.19 +/-0.20 mmol/L (mean +/- standard error), the activation energy (Ea) was 108.3+/-1.2 kJ/ mol, and the residual enzyme activity after heating at 45 degrees C for 20 min was 79.6+/-1.9%. On the other hand, octameric uMi-CK was focused at pI 7.1-7.9 and the dimeric forms were focused at pI 6.6, 6.7, 6.8, 6.9, and 7.0. Delysined forms were focused around pI 6.3, 6.4, 6.8, and 6.9. uMi-CK reacted with anti-sMi-CK antibodies, and the immune complexes were focused at pI 5.8. The Km value of uMi-CK for PCr was 1.07+/-0.03 mmol/L, Ea of uMi-CK was 110.0+/-0.9 kJ/mol, and the residual enzyme activity after heating at 45 degrees C for 20 min was 90.3+/-0.4%. The sMi-CK and uMi-CK were hybridized and the hybrid Mi-CK appeared at pI 6.78, 6.98, and 7.1-7.95. The pIs of the hybrid Mi-CK were between those of sMi-CK and uMi-CK. As described above, sMi-CK and uMi-CK were slightly different from each other with respect to the pI and some enzyme characteristics.     

Heats of formation of the propionyl ion and radical and 2,3-pentanedione by threshold photoelectron photoion coincidence spectroscopy

The dissociative photoionization onsets for the formation of the propionyl ion (C(2)H(5)CO(+)) and the acetyl ion (CH(3)CO(+)) were measured from energy selected butanone and 2,3-pentanedione ions using the technique of threshold photoelectron photoion coincidence (TPEPICO) spectroscopy. Ion time-of-flight (TOF) mass spectra recorded as a function of the ion internal energy permitted the construction of breakdown diagrams, which are the fractional abundances of ions as a function of the photon energy. The fitting of these diagrams with the statistical theory of unimolecular decay permitted the extraction of the 0 K dissociation limits of the first and second dissociation channels. This procedure was tested using the known energetics of the higher energy dissociation channel in butanone that produced the acetyl ion and the ethyl radical. By combining the measured dissociative photoionization onsets with the well-established heats of formation of CH(3)(*), CH(3)CO(+), CH(3)CO(*), and butanone, the 298 K heats of formation, Delta(f)H degrees (298K), of the propionyl ion and radical were determined to be 618.6 +/- 1.4 and -31.7 +/- 3.4 kJ/mol, respectively, and Delta(f)H degrees (298K)[2,3-pentanedione] was determined to be -343.7 +/- 2.5 kJ/mol. This is the first experimentally determined value for the heat of formation for 2,3-pentanedione. Ab initio calculations at the Weizmann-1 (W1) level of theory predict Delta(f)H degrees (298K) values for the propionyl ion and radical of 617.9 and -33.3 kJ/mol, respectively, in excellent agreement with the measured values.     

Helium atom scattering study of the interaction of water with the BaF2(111) surface

The interaction of water with the BaF2(111) single crystal surface is investigated using the helium atom scattering technique. It is found that H2O forms a long-range ordered two-dimensional (2D) phase with (1 x 1) translational symmetry already after an exposure of 3 L (1 L=10(-6) Torr s) at temperatures below 150 K. The activation energy for desorption of the saturated 2D phase, which is assigned to a bilayer, is estimated to be 46+/-2 kJ mol(-1), corresponding to a desorption temperature of 165 K. The desorption of multilayers was observed at 150 K, consistent with a binding energy of 42+/-2 kJ mol(-1). Before completion and after desorption of the saturated 2D phase, a superstructure consistent with a disordered (square root of 3 x square root of 3)R30 degrees lattice has been observed, which is attributed to the first layer of water with a coverage of one molecule per surface unit cell, in accordance with recent theoretical studies. Desorption of this phase is observed at temperatures above 200 K, consistent with an unexpectedly strong bonding of the molecules to the substrate.     

Dissociation dynamics of sequential ionic reactions: heats of formation of tri-, di-, and monoethylphosphine

The sequential ethene (C2H4) loss channels of energy-selected ethylphosphine ions have been studied using threshold photoelectron photoion coincidence (TPEPICO) spectroscopy in which ion time-of-flight (TOF) distributions are recorded as a function of the photon energy. The ion TOF distributions and breakdown diagrams have been modeled in terms of the statistical RRKM theory for unimolecular reactions, providing 0 K dissociation onsets, E0, for the ethene loss channels. Three RRKM curves were used to model the five measurements, since two of the reactions differ only by the internal energy of the parent ion. This series of dissociations provides a detailed check of the calculation of the product energy distribution for sequential reactions. From the determined E0's, the heats of formation of several ethylphosphine neutrals and ions have been determined: Delta(f)H degrees 298K[P(C(2)H(5))3] = -152.7 +/- 2.8 kJ/mol, Delta(f)H degrees 298K[P(C(2)H(5))3+] = 571.6 +/- 4.0 kJ/mol, Delta(f)H degrees 298K[HP(C(2)H(5))2] = -89.6 +/- 2.1 kJ/mol, Delta(f)H degrees 298K[HP(C(2)H(5))2+] = 669.9 +/- 2.5 kJ/mol, Delta(f)H degrees 298K[H(2)PC(2)H(5)] = -36.5 +/- 1.5 kJ/mol, Delta(f)H degrees 298K[H(2)PC(2)H(5)+] = 784.0 +/- 1.9 kJ/mol. These values have been supported by G2 and G3 calculations using isodesmic reactions. Coupled cluster calculations have been used to show that the C2H4 loss channel, which involves a hydrogen transfer step, proceeds without a reverse energy barrier.