Hemin (Fe(3+))-- and heme (Fe(2+))--smectite conjugates as a model of hemoprotein based on spectrophotometry

Hemin (Fe(3+)) was adsorbed onto synthetic smectite (clay mineral) in acetone to form a hemin-smectite conjugate. The hemin-smectite conjugate became soluble in water to form a transparent colloidal solution with a dark brown color. Its absorption spectrum in water showed a sharp Soret band at 398 nm with the molar extinction coefficient as epsilon(398nm) = 11.6 x 10(4) M(-1) cm(-1), which is in good agreement with epsilon(398nm) = (12.2 +/- 3) x 10(4) M(-1) cm(-1) of monomeric hematin (1). Hemin (Fe(3+))-smectite conjugate had a peroxidase-like activity in the presence of hydrogen peroxide (a hydrogen acceptor) and guaiacol (a hydrogen donor) in aqueous solution and its activity was higher than that of hematin. Hemin (Fe(3+))-smectite conjugate in water was reduced by adding sodium dithionite to form a heme (Fe(2+))-smectite conjugate which is also a transparent colloidal solution in water. Its absorption spectrum in aqueous solution was surprisingly in close agreement with that of oxyhemoglobin. Its peak positions of alpha, beta, and Soret bands were located in only a 9--3 nm shift to shorter wavelengths in comparison with those of oxyhemoglobin. Therefore, heme (Fe(2+))-smectite conjugate was bound to O(2) to form O(2)-heme (Fe(2+))-smectite conjugate. The addition of carbon monoxide, CO, to O(2)-heme (Fe(2+))-smectite conjugate caused the formation of CO-heme (Fe(2+))-smectite conjugate with a similar absorption spectrum of carboxyhemoglobin (HbCO) accompanied by shifting 8--10 nm to shorter wavelength. Therefore, the transformation of O(2)-heme (Fe(2+))-smectite conjugate to CO-heme (Fe(2+))-smectite conjugate was accompanied by shifting of 7, 4, and 3 nm to shorter wavelengths in the alpha, beta, and Soret bands respectively, which are similar to the spectral change from oxyhemoglobin to carboxyhemoglobin. Also the ratio (1:1.6) of the molar extinction coefficient of Soret band of O(2)-heme (Fe(2+))-smectite conjugate and CO-heme (Fe(2+))-smectite conjugate was surprisingly agreement with ratio (1:1.5) of oxyhemoglobin and carboxyhemoglobin. The phenomenon shown above was unexpectedly found during the course of study of bioconjugate of a bioactive substance, hemin (Fe(3+)) or heme (Fe(2+)), and a clay mineral, smectite, in place of the protein of globin in hemoglobin.     

Hemoprotein models based on a covalent helix-heme-helix sandwich. 3. Coordination properties, reactivity and catalytic application of Fe(III)- and Fe(II)-mimochrome I

 The coordination state of Fe(III)- and Fe(II)-mimochrome I, a covalent peptide-deuteroheme sandwich involving two nonapeptides bearing a histidine residue in a central position, was studied by UV-visible, EPR, and resonance Raman spectroscopy. The ferric and ferrous states of this new iron species mainly exist, at pH 7, in a low-spin hexacoordinate form with two axial histidine ligands coming from the peptide chains. A minor amount of high-spin form for the ferric state is also present at pH 7. However, it is mainly high-spin at pH 2 or in DMSO. Fe(II)-mimochrome I binds CO with an affinity comparable to that of myoglobin and hemoglobin. Fe(III)-mimochrome I reacts with alkylhydroxylamine and arylhydrazines, leading to the corresponding Fe(II)-nitrosoalkyl and Fe(III)-σ-aryl complexes, respectively. These reactions were greatly dependent on the solvent used and on the pH, and were much slower than the corresponding reactions performed by deuterohemin in the presence of excess imidazole. All these results indicate that the reactivity of iron-mimochrome I is controlled by the binding of the peptide chains to the iron. The reactivity shown by this complex at neutral pH is intermediate between that observed for iron porphyrins in the presence of excess imidazole and that of hemoproteins characterized by a strong bis-histidine axial coordination, such as cytochrome b ₅. Fe(III)-mimochrome I is able to catalyze styrene epoxidation by using a [Fe(III)-mimochrome I]/[H₂O₂]/[stryrene] ratio of 1 : 10 : 2000 in phosphate buffer solution (pH 7.2) containing 2% CTAB both under strictly anaerobic conditions and in the presence of oxygen, at 0  °C. Received: 26 May 1998 / Accepted: 20 August 1998     

Absorption of Iron-Dextrin (Fe59) and Iron-Sorbitol (Fe59) from an I. M. Injection in Piglets

Absorption of iron has been followed after the intramuscular injection of 12 piglets with iron-dextrin and iron-sorbitol, labelled with Fe59. Disappearance from the site of injection was followed by a method developed for external radioactivity measurements. Blood levels of Fe59 were measured at intervals during a period of 51 days. The tissue distribution pattern of Fe59 was examined in six of the piglets after 30 days. Both iron-dextrin and iron-sorbitol were rapidly absorbed, iron-sorbitol more rapidly. After 20 minutes, 50 per cent of the iron-sorbitol had been absorbed and less than one per cent remained at the site of injection after 48 hours. The amount of Fe59 remaining at the site of injection of iron-dextrin had decreased by half by 60 minutes but some ten per cent had still not been absorbed after ten days. When given as iron-dextrin (94 mg Fe per piglet), the level of Fe59 in the blood remained fairly constant for 14 days. On piglets given iron-sorbitol, (100 mg Fe each) the blood level declined steadily. The amount of Fe59 in the liver, spleen and bone marrow was much greater 30 days after the injection of iron-dextrin than after iron-sorbitol. The relation was reversed in the kidneys, presumably because of the greater excretion of iron-sorbitol in the urine.     

Deformability of Fe(II)CO and Fe(III)CN groups in heme protein models: nonlocal density functional theory calculations

 High-quality nonlocal density functional calculations have been carried out on the deformability of Fe(II)CO and Fe(III)CN units in model compounds of heme proteins. The results confirm our previous finding with a local functional that the Fe(II)CO unit is significantly deformable with respect to tilting and bending. This deformability stems in large part from a large, negative interaction constant between the FeC tilt and FeCO bend coordinates. The Fe(III)CN unit is also significantly deformable, but in this case the deformability results from a very small Fe(III)CN bend force constant and the ability of the cyano nitrogen to act as a hydrogen bond acceptor. The prediction that the energetic penalty associated with deforming the Fe(II)CO unit is relatively modest indicates that such deformations are unlikely to be the dominant contributor to myoglobin's discrimation against CO in favor of O₂. Received, accepted: 16 June 1997     

Simple computational models of type I/type II cells in Fas signaling-induced apoptosis

Distinct apoptotic response of the type I/type II cells against Fas-ligand stimulation is considered to arise from the difference in dominant signaling pathways involved. In the type I cells, apoptotic signaling predominantly takes place via the direct activation of caspase-3 by activated caspase-8 (D channel) while mitochondrial pathway (M channel) plays a major role in the type II cells. To elucidate the selection mechanism of dominant pathway, we carried out systematic model analysis of the Fas signaling-induced apoptosis network. An increase in the expression level of caspase-8 induced a switch of dominant pathway from M- to D-channel (M–D transition), showing a phenotypic change from type II to type I cells. With the aid of sensitivity analysis and kinetic considerations, we succeeded in constructing a minimal network model relevant for the M–D transition, which revealed that mechanistic origin of the transition lies in the competition between the activated forms of caspase-8 and caspase-9 for their common substrate caspase-3. The pathway dominance was found to be primarily controlled by the balance between the activation rate of caspase-8 and the initial level of caspase-9. In the full network model, we showed that differential formation ability of the death-inducing signaling complex (DISC) can also induce M–D transition, in accordance with the experimental observations.     

A biological approach to computational models of proteomic networks

Computational modeling is useful as a means to assemble and test what we know about proteins and networks. Models can help address key questions about the measurement, definition and function of proteomic networks. Here, we place these biological questions at the forefront in reviewing the computational strategies that are available to analyze proteomic networks. Recent examples illustrate how models can extract more information from proteomic data, test possible interactions between network proteins and link networks to cellular behavior. No single model can achieve all these goals, however, which is why it is critical to prioritize biological questions before specifying a particular modeling approach.     

Molecular dynamics simulations of solvated crystal models of cellulose I(alpha) and III(I)

Swelling behaviors of cellulose I(alpha) and III(I) crystals have been studied using molecular dynamics simulations of the solvated finite-crystal models. The typical crystal models consisted of 48 x 10-mer chains. For the cellulose I(alpha) crystal, models consisting of different numbers of chains and chain lengths were also studied. The structural features of the swollen crystal models, including the cellulose I(beta) crystal model reported previously, were compared. A distinct right-handed twist was observed for models of the native cellulose crystals (cellulose I(alpha) and I(beta)), with a greater amount of twisting observed for the I(alpha) crystal model. Although the amount of twist decreased with increasing dimensions of the cellulose I(alpha) crystal model, the relative changes in twist angle suggest that considerable twist would arise in a crystal model of the actual dimensions. In contrast to the swelling behavior of crystal models of the native cellulose, the cellulose III(I) crystal model exhibited local, gradual disordering at the corner of the reducing end. Comparison of the lattice energies indicated that the cellulose chains of the I(beta) crystal were packed in the most stable fashion, whereas those of the I(alpha) and III(I) crystals were in a metastable state, which is consistent with the crystallization behaviors observed. Upon heating of the native cellulose crystal models, the chain sheets of the I(alpha) model showed a continuous increase in twist angle, suggesting weaker intersheet interactions in this model. The swollen crystal models of cellulose I(alpha) and III(I) reproduce well the representative structural features observed in the corresponding crystal structures. The crystal model twist thus characterizes the swelling behavior of the native cellulose crystal models, which seems to be related to the insolubility of the crystals.     

A computational modeling and analysis in cell biological dynamics using electric cell-substrate impedance sensing (ECIS)

In this paper, a study of computational modeling and multi-scale analysis in cell dynamics is presented. Our study aims at: (1) deriving and validating a mathematical model for cell growth, and (2) quantitatively detecting and analyzing the biological interdependencies across multiple observational scales with a variety of time and frequency resolutions. This research was conducted using the time series data practically measured from a novel on-line cell monitoring technique, referred to as electric cell-substrate impedance sensing (ECIS), which allows continuously tracking the cellular behavior such as adhesion, proliferation, spreading and micromotion. First, comparing our ECIS-based cellular growth modeling analysis results with those determined by hematocytometer measurement using different time intervals, we found that the results obtained from both experimental methods consistently agreed. However, our study demonstrated that it is much easier and more convenient to operate with the ECIS system for on-line cellular growth monitoring. Secondly, for multi-scale analysis our results showed that the proposed wavelet-based methodology can effectively quantify the fluctuations associated with cell micromotions and quantitatively capture the biological interdependencies across multiple observational scales. Note that although the wavelet method is well known, its application into the ECIS time series analysis is novel and unprecedented in computational cell biology. Our analyses indicated that the proposed study on ECIS time series could provide a hopeful start and great potentials in both modeling and elucidating the complex mechanisms of cell biological systems.     

Model-free analysis of a thermophilic Fe(7)S(8) protein compared with a mesophilic Fe(4)S(4) protein

15N T(1), T(2) and (1)H-(15)N NOE were measured for the thermophilic Fe(7)S(8) protein from Bacillus schlegelii and for the Fe(4)S(4) HiPIP protein from Chromatium vinosum, which is a mesophilic protein. The investigation was performed at 276, 300, and 330 K at 11.7 T for the former, whereas only the 298 K data at 14.1 T for the latter were acquired. The data were analyzed with the model-free protocol after correcting the measured parameters for the effect of paramagnetism, because both proteins are paramagnetic. Both thermophilic and mesophilic proteins are quite rigid, with an average value of the generalized order parameter S2at room temperature of 0.92 and 0.94 for Fe(7)S(8) and Fe(4)S(4) proteins, respectively. The analyzed nitrogens for the Fe(7)S(8) protein showed a significant decrease in S2with increasing temperature, and at the highest temperature >70% of the residues had an internal correlation time. This research shows that subnanosecond rigidity is not related to thermostability and provides an estimate of the effect of increasing temperature on this time scale.     

A comparison of two procedures used for complexing Fe(III) with human apotransferrin: I. Physicochemical properties of the Fe(III) . transferrin products

Samples of human Fe.transferrin (Fe.HTr) were prepared from a single batch of apotransferrin (apo.HTr) by either the Fe(III)-citrate or the Fe(II)-ceruloplasmin (ferroxidase) method. By using 55Fe, 55Fe.HTr prepared by the citrate method and 55Fe.HTr prepared by the ceruloplasmin method contained 2.2-2.3 and 2.0 Fe/mol, respectively. For both 55Fe.HTr preparations, the isotope was shown to be associated with the protein from the measurement of absorbance at 465 nm and dialysis studies. However, passage of the 55Fe.HTr (ceruloplasmin) reaction mixture through DEAE-cellulose caused 55-60% of 55Fe to be lost from the protein, although no decrease in absorbance at 465 nm was observed. Ion-exchange chromatography of 55Fe.HTr (citrate) did not induce loss of 55Fe. Absorbance measurements showed significant differences between the two Fe.HTr preparations with respect to the ratios A212/A278 and A463/A278. Using an excitation wavelength of 275 nm, the fluorescence intensity ratios relative to apo.HTr were 0.275 and 0.309 for Fe.HTr (citrate) and Fe.HTr (ceruloplasmin), respectively. Electron spin resonance (ESR) measurements confirmed that Fe.HTr (citrate) and Fe.HTr (ceruloplasmin) were saturated with Fe. Hyperfine coupling constants and other features of the resonance profile revealed distinct differences between the two Fe.HTr preparations. Dialysis against H2O caused Fe.HTr (citrate), but not Fe.HTr (ceruloplasmin), to lose absorbance at 465 nm. The ESR profile of Fe.HTr (citrate), after dialysis against H2O, was reduced to multiple splittings and a lack of resolution of the central hyperfine structure. Addition of Na2CO3 restored the absorbance (465 nm) and the ESR pattern of Fe.HTr (citrate). In contrast, these properties of Fe.HTr (ceruloplasmin) were little affected by dialysis against H2O. However, the addition of trisodium citrate to Fe.HTr (ceruloplasmin) caused a reduction in absorbance at 465 nm and a change in ESR profile to resemble that of Fe.HTr (citrate) after dialysis in H2O; these changes, caused by citrate binding to Fe.HTr (ceruloplasmin), were restored to normal by the addition of Na2CO3. The data indicate that different protein conformations result from complexing Fe(III) with apo.HTr by these two different procedures. The two Fe.HTr products may differ, conceivably, in their abilities to transfer Fe to cells.     

A computational comparison of the atomic models of the actomyosin interface

Several atomic models of the actomyosin interface have been proposed based on the docking together of their component structures using electron microscopy and resonance energy-transfer measurements. Although these models are in approximate agreement in the location of the binding interfaces when myosin is tightly bound to actin, their relationships to molecular docking simulations based on computational free-energy calculations are investigated here. Both rigid-docking and flexible-docking conformational search strategies were used to identify free-energy minima at the interfaces between atomic models of myosin and actin. These results suggest that the docking model produced by resonance energy-transfer data is closer to a free-energy minimum at the interface than are the available atomic models based on electron microscopy. The conformational searches were performed using both scallop and chicken skeletal muscle myosins and identified similarly oriented actin-binding interfaces that serve to validate that these models are at the global minimum. These results indicate that the existing docking models are close to but not precisely at the lowest-energy initial contact site for strong binding between myosin and actin that should represent an initial contact between the two proteins; therefore, conformational changes are likely to be important during the transition to a strongly bound complex.     

The effective molarity (EM) - A computational approach

The effective molarity (EM) for 12 intramolecular S_N2 processes involving the formation of substituted aziridines and substituted epoxides were computed using ab initio and DFT calculation methods. Strong correlation was found between the calculated effective molarity and the experimentally determined values. This result could open a door for obtaining EM values for intramolecular processes that are difficult to be experimentally provided. Furthermore, the calculation results reveal that the driving forces for ring-closing reactions in the two different systems are proximity orientation of the nucleophile to the electrophile and the ground strain energies of the products and the reactants.     

A computational model for previtamin D(3) production in skin

Low levels of vitamin D have been implicated in a wide variety of health issues from calcemic diseases to cancer, diabetes and cardiovascular disease. For most humans, the majority of vitamin D(3) is derived from sunlight. How much vitamin D is produced under given exposure conditions is still widely discussed. We present a computational model for the production of (pre-)vitamin D within the skin. It accounts for spectral irradiance, optical properties of the skin and concentration profile of provitamin D. Results are computed for various sets of these parameters yielding the distribution of produced previtamin D in the skin.     

Computational definition of a mixed valent Fe(II)Fe(I) model of the [FeFe]hydrogenase active site resting state

Density-functional calculations have been used to examine the electronic structure and bonding in the recently reported complex [(PMe(3))(CO)(2)Fe(mu-pdt)(mu-CO)Fe(CO)(IMes)](+) (1(+), IMes=1,3-bis(2,4,6-trimethylphenyl)-imidazol-2-ylidene). This mixed valent Fe(II)Fe(I) complex features a rotated geometry that places a carbonyl ligand in a semi-bridging position, which makes it an accurate model of the S =(1/2) resting state of the [FeFe]-hydrogenase active site. Calculations indicate that the unpaired electron in this complex lies almost entirely on the rotated iron center, implying that this iron remains in the Fe(I) oxidation state, while the unrotated iron has been oxidized to Fe(II). The frontier molecular orbitals in 1(+) are compared with those in the neutral Fe(I)Fe(I) precursor (PMe(3))(CO)(2)Fe(mu-pdt)(mu-CO)Fe(CO)(IMes) at both its optimized geometry (1) and constrained to a rotated geometry (1(rot)). These theoretical results are used to address the role of the bridging CO ligand in 1(+) and to predict reactivity patterns; they are related back to the intricate biological mechanism of [FeFe]-hydrogenase.     

The dynamics of (57)Fe nuclei in Fe(III)-DNA condensates.

The dynamics of iron nuclei in the condensates obtained by interaction of Fe(III) with DNA, Fe(III)(DNA monomer)(2), have been investigated by variable temperature (57)Fe M?ssbauer spectroscopy. Studies were effected on gel and freeze-dried samples, obtaining nearly coincident values of the parameters isomer shift and nuclear quadrupole splitting in T ranges 20-260 K. Functions ln(A(T)/A(77.3)) vs. T, here employed to investigate the dynamics of Fe nuclei, showed linear trends in the T ranges 20-150 and 150-260 K, respectively, the latter with larger slopes. Data coincided for gelled and freeze-dried specimens. No variation of delta or Delta E parameters occurred at the two T intervals, which suggests constancy of structure and bonding with the temperature changes. Functions (T) showed trends analogous to the corresponding functions determined for iron proteins, which were attributed to the occurrence of 'conformational substates'.     

A computational image analysis glossary for biologists

Recent advances in biological imaging have resulted in an explosion in the quality and quantity of images obtained in a digital format. Developmental biologists are increasingly acquiring beautiful and complex images, thus creating vast image datasets. In the past, patterns in image data have been detected by the human eye. Larger datasets, however, necessitate high-throughput objective analysis tools to computationally extract quantitative information from the images. These tools have been developed in collaborations between biologists, computer scientists, mathematicians and physicists. In this Primer we present a glossary of image analysis terms to aid biologists and briefly discuss the importance of robust image analysis in developmental studies.     

A new computational efficient approach for trabecular bone analysis using beam models generated with skeletonized graph technique

Micro-finite element (FE) analysis is a well established technique for the evaluation of the elastic properties of trabecular bone, but is limited in its application due to the large number of elements that it requires to represent the complex internal structure of the bone. In this paper, we present an alternative FE approach that makes use of a recently developed 3D-Line Skeleton Graph Analysis (LSGA) technique to represent the complex internal structure of trabecular bone as a network of simple straight beam elements in which the beams are assigned geometrical properties of the trabeculae that they represent. Since an enormous reduction of cputime can be obtained with this beam modeling approach, ranging from approximately 1,200 to 3,600 for the problems investigated here, we think that the FE modeling technique that we introduced could potentially constitute an interesting alternative for the evaluation of the elastic mechanical properties of trabecular bone.     

A computational model of antibiotic-resistance mechanisms in methicillin-resistant Staphylococcus aureus (MRSA)

An agent-based model of bacteria-antibiotic interactions has been developed that incorporates the antibiotic-resistance mechanisms of Methicillin-Resistant Staphylococcus aureus (MRSA). The model, called the Micro-Gen Bacterial Simulator, uses information about the cell biology of bacteria to produce global information about population growth in different environmental conditions. It facilitates a detailed systems-level investigation of the dynamics involved in bacteria-antibiotic interactions and a means to relate this information to traditional high-level properties such as the Minimum Inhibitory Concentration (MIC) of an antibiotic. The two main resistance strategies against β-lactam antibiotics employed by MRSA were incorporated into the model: β-lactamase enzymes, which hydrolytically cleave antibiotic molecules, and penicillin-binding proteins (PBP2a) with reduced binding affinities for antibiotics. Initial tests with three common antibiotics (penicillin, ampicillin and cephalothin) indicate that the model can be used to generate quantitatively accurate predictions of MICs for antibiotics against different strains of MRSA from basic cellular and biochemical information. Furthermore, by varying key parameters in the model, the relative impact of different kinetic parameters associated with the two resistance mechanisms to β-lactam antibiotics on cell survival in the presence of antibiotics was investigated.     

A condition for setting off ectopic waves in computational models of excitable cells

The purpose of this paper is to study the stability of steady state solutions of the Monodomain model equipped with Luo-Rudy I kinetics. It is well established that re-entrant arrhythmias can be created in computational models of excitable cells. Such arrhythmias can be initiated by applying an external stimulus that interacts with a partially refractory region, and spawn breaking waves that can eventually generate extremely complex wave patterns commonly referred to as fibrillation. An ectopic wave is one possible stimulus that may initiate fibrillation. Physiologically, it is well known that ectopic waves exist, but the mechanism for initiating ectopic waves in a large collection of cells is poorly understood. In the present paper we consider computational models of collections of excitable cells in one and two spatial dimensions. The cells are modeled by Luo-Rudy I kinetics, and we assume that the spatial dynamics is governed by the Monodomain model. The mathematical analysis is carried out for a reduced model that is known to provide good approximations of the initial phase of solutions of the Luo-Rudy I model. A further simplification is also introduced to motivate and explain the results for the more complicated models. In the analysis the cells are divided into two regions; one region (N) consists of normal cells as model by the standard Luo-Rudy I model, and another region (A) where the cells are automatic in the sense that they would act as pacemaker cells if they where isolated from their surroundings. We let delta denote the spatial diffusion and a denote a characteristic length of the automatic region. It has previously been shown that reducing diffusion or increasing the automatic region enhances ectopic activity. Here we derive a condition for the transition from stable resting state to ectopic wave spread. Under suitable assumptions on the model we provide mathematical and computational arguments indicating that there is a constant eta such that a steady state solution of this system is stable whenever delta approximately > etaa(2), and unstable whenever delta approximately < etaa(2).