RESUMO
The specific delivery of chemotherapeutic agents to their desired targets with a minimum of systemic side effects is an important, ongoing challenge of chemotherapy. One approach, developed in the past to address this problem, is the i.v. injection of magnetic particles [ferrofluids (FFs)] bound to anticancer agents that are then concentrated in the desired area (e.g., the tumor) by an external magnetic field. In the present study, we treated squamous cell carcinoma in rabbits with FFs bound to mitoxantrone (FF-MTX) that was concentrated with a magnetic field. Experimental VX-2 squamous cell carcinoma was implanted in the median portion of the hind limb of New Zealand White rabbits (n = 26). When the tumor had reached a volume of approximately 3500 mm3, FF-MTX was injected intraarterially (i.a.; femoral artery) or i.v. (ear vein), whereas an external magnetic field was focused on the tumor. FF-MTX i.a. application with the external magnetic field resulted in a significant (P < 0.05), complete, and permanent remission of the squamous cell carcinoma compared with the control group (no treatment) and the i.v. FF-MTX group, with no signs of toxicity. The intratumoral accumulation of FFs was visualized both histologically and by magnetic resonance imaging. Thus, our data show that i.a. application of FF-MTX is successful in treating experimental squamous cell carcinoma. This "magnetic drug targeting" offers a unique opportunity to treat malignant tumors locoregionally without systemic toxicity. Furthermore, it may be possible to use these magnetic particles as a "carrier system" for a variety of anticancer agents, e.g., radionuclides, cancer-specific antibodies, and genes.
Assuntos
Antineoplásicos/administração & dosagem , Sistemas de Liberação de Medicamentos , Magnetismo , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Animais , Antineoplásicos/farmacocinética , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Coloides/administração & dosagem , Coloides/farmacocinética , Portadores de Fármacos , Feminino , Compostos Férricos/administração & dosagem , Compostos Férricos/farmacocinética , Imageamento por Ressonância Magnética , Mitoxantrona/administração & dosagem , Mitoxantrona/farmacocinética , Neoplasias Experimentais/patologia , CoelhosRESUMO
The phonon-assisted Mössbauer effect is used to determine the partial phonon density of states of the iron within the active center of deoxymyoglobin, carboxymyoglobin, and dry and wet metmyoglobin between 40 and 300 K. Between 0 and 1 meV the iron density of states increases quadratically with the energy, as in a Debye solid. Mean sound velocities are extracted from this slope. Between 1 and 3 meV a nearly quadratic "Debye-like" increase follows due to the similar strength of intermolecular and intramolecular forces. Above 3 meV, optical vibrations are characteristic for the iron-ligand conformation. The overall mean square displacements of the heme iron atom obtained from the density of states agree well with the values of Mössbauer absorption experiments below 180 K. In the physiological temperature regime the data confirm the existence of harmonic vibrations in addition to the protein specific dynamics measured by Mössbauer absorption. In the Debye energy regime the mean square displacement of the iron is in agreement with that of the hydrogens measured by incoherent neutron scattering demonstrating the global character of these modes. At higher energies the vibration of the heavy iron atom at 33 meV in metmyoglobin is as large as that of the lightweight hydrogens at that energy. A freeze dried, rehydrated (h=0.38 g H2O/g protein) metmyoglobin sample shows an excess of states above the Debye law between 1 and 3 meV, similar to neutron scattering experiments. The room temperature density of states below 3 meV exhibit an increase of the density compared to the low temperature data, which can be interpreted as mode softening.
RESUMO
A frozen solution of 57Fe-enriched metmyoglobin was irradiated by x rays at 77 K. Mössbauer spectra showed a reduction of Fe(III) high spin by thermalized electrons and a production of a metastable Fe(II) low spin myoglobin complex with H2O at its sixth coordination site. The relaxation of the intermediate was investigated by Mössbauer spectroscopy as a function of temperature and time. The relaxation process starts above 140 K and is fully completed at approximately 200 K. At temperatures between 140 and 200 K, the relaxation lasts for hours and is nonexponential in time. Up to 180 K, the process can be described satisfactorily by a gamma distribution of activation enthalpies with an Arrhenius relation for the rate coefficient. The temperature and time dependence of the Mössbauer parameters indicates structural changes in the active center of the protein as early as 109 K that continue for several hours at higher temperatures. Above 180 K, structural rearrangements involving the whole protein molecule lead to a shift and narrowing of the barrier height distribution.
Assuntos
Mioglobina/química , Conformação Proteica , Animais , Isótopos de Ferro , Cinética , Matemática , Metamioglobina/química , Modelos Teóricos , Mioglobina/efeitos da radiação , Espectroscopia de Mossbauer/métodos , Termodinâmica , Baleias , Raios XRESUMO
Protein motions are essential for function. Comparing protein processes with the dielectric fluctuations of the surrounding solvent shows that they fall into two classes: nonslaved and slaved. Nonslaved processes are independent of the solvent motions; their rates are determined by the protein conformation and vibrational dynamics. Slaved processes are tightly coupled to the solvent; their rates have approximately the same temperature dependence as the rate of the solvent fluctuations, but they are smaller. Because the temperature dependence is determined by the activation enthalpy, we propose that the solvent is responsible for the activation enthalpy, whereas the protein and the hydration shell control the activation entropy through the energy landscape. Bond formation is the prototype of nonslaved processes; opening and closing of channels are quintessential slaved motions. The prevalence of slaved motions highlights the importance of the environment in cells and membranes for the function of proteins.
Assuntos
Proteínas/fisiologia , Transferência de Energia , Modelos Químicos , Movimento (Física) , Conformação Proteica , Proteínas/química , Solubilidade , Soluções , Solventes , Relação Estrutura-Atividade , Temperatura , VibraçãoRESUMO
Myoglobin, a small globular haem protein that binds gaseous ligands such as O2, CO and NO reversibly at the haem iron, serves as a model for studying structural and dynamic aspects of protein reactions. Time-resolved spectroscopic measurements after photodissociation of the ligand revealed a complex ligand-binding reaction with multiple kinetic intermediates, resulting from protein relaxation and movements of the ligand within the protein. To observe the structural changes induced by ligand dissociation, we have carried out X-ray crystallographic investigations of carbon monoxy-myoglobin (MbCO mutant L29W) crystals illuminated below and above 180 K, complemented by time-resolved infrared spectroscopy of CO rebinding. Here we show that below 180 K photodissociated ligands migrate to specific sites within an internal cavity--the distal haem pocket--of an essentially immobilized, frozen protein, from where they subsequently rebind by thermally activated barrier crossing. Upon photodissociation above 180 K, ligands escape from the distal pocket, aided by protein fluctuations that transiently open exit channels. We recover most of the ligands in a cavity on the opposite side of the haem group.
Assuntos
Mioglobina/metabolismo , Animais , Cristalografia por Raios X , Escherichia coli , Heme/química , Heme/metabolismo , Cinética , Ligantes , Mioglobina/química , Fotólise , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrofotometria Infravermelho , Baleias , Xenônio/químicaRESUMO
Metmyoglobin has been reduced at low temperature (below 100 K) using x-rays or by excitation of tris(2,2,bipyridine)ruthenium(II) chloride with visible light. Upon reduction, an intermediate state is formed where the structure of the protein is very similar to that of metmyoglobin with the water molecule still bound to the heme iron, but the iron is II low spin. The nature of the intermediate state has been investigated with optical spectroscopy. The Q(O) and Q(V) bands of the intermediate state are split, suggesting that the protoporphyrin is distorted. The intermediate state undergoes a relaxation observed by a shifting of the Soret band at temperatures above 80 K. Above 140 K, the protein begins to relax to the deoxy conformation. The relaxation kinetics of the protein have been monitored optically as a function of time and temperature from minutes to several hours and from 150 K to 190 K. By measuring the entire visible spectrum, we are able to distinguish between electron transfer processes and the protein relaxation from the intermediate state to deoxy myoglobin. The relaxation has been measured in both horse myoglobin and sperm whale myoglobin with the relaxation occurring on faster time scales in horse myoglobin. Both the reduction kinetics and the relaxation show non-exponential behavior. The reduction kinetics can be fit well to a stretched exponential. The structural relaxation from the intermediate state to the deoxy conformation shows a more complex, dynamical behavior and the reaction is most likely affected by the relaxation of the protein within the intermediate state.
Assuntos
Metamioglobina/química , Mioglobina/análogos & derivados , 2,2'-Dipiridil/análogos & derivados , 2,2'-Dipiridil/química , Animais , Temperatura Baixa , Complexos de Coordenação , Compostos Ferrosos/química , Indicadores e Reagentes/química , Cinética , Mioglobina/química , Oxirredução , Fotoquímica , Conformação Proteica , Espectrofotometria , BaleiasRESUMO
Rats have been enriched in 57Fe and erythrocytes were isolated from the blood. Mössbauer absorption spectroscopy on the hemoglobin of these erythrocytes has shown rather similar dynamics as found earlier in crystals of myoglobin, in frozen solutions of human hemoglobin and in a number of other proteins. The results strongly indicate that the motion of the heme and presumably some part of the F-helix is mainly influenced by the average viscosity of the sample determined by a network of hydrogen bridges and other weak interactions. Extrapolations of Mössbauer results from protein crystals to proteins in their physiological surroundings seem to be suitable for heme proteins.
Assuntos
Eritrócitos/metabolismo , Hemoglobinas/metabolismo , Animais , Heme/metabolismo , Hemoglobinas/química , Ferro/metabolismo , Cinética , Conformação Proteica , Ratos , Espectroscopia de MossbauerRESUMO
Mössbauer, 57Fe ENDOR, CW and pulsed EPR experiments were performed on the reduced and the oxidized high-potential iron proteins (HiPIPs) of the wild type (WT) and the C77S mutant from Chromatium vinosum. The EPR spectra of the oxidized WT and mutant show three species respectively having nearly the same g-values but strongly changed spectral contributions. Relaxation times were estimated for oxidized WT and mutant at T = 5 K with pulsed EPR. A-tensor components of both iron pairs were obtained by 57Fe ENDOR, proving a similar magnetic structure for the WT and the mutant. Electronic relaxation has to be taken into account at T = 5 K in native and mutated oxidized HiPIPs to achieve agreement between Mössbauer and 57Fe ENDOR spectroscopies. The Mössbauer spectroscopy shows that the oxidized cluster contains a pure ferric and a mixed-valence iron pair coupled antiparallel. While all cluster irons from reduced C. vinosum WT are indistinguishable in the Mössbauer spectrum, the reduced C77S mutant shows a non-equivalence between the serine-bound and the three cysteine-ligated iron ions. The Mössbauer parameters confirm a loss of the covalent character of the iron bond when S is replaced by O and indicate a shift of the cluster's electron cloud towards the serine. Mössbauer spectra of the oxidized mutant can be simulated with two models: model I introduces a single electronic isomer with the serine always ligated to a ferric iron. Model II assumes two equally populated electronic isomers with the serine ligated to a ferric iron and a mixed-valence iron, respectively. The latter model is in better agreement with EPR and NMR.
Assuntos
Chromatium/química , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/metabolismo , Mutação , Complexo de Proteínas do Centro de Reação Fotossintética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Proteínas Ferro-Enxofre/genética , Isomerismo , Magnetismo , Estrutura Molecular , Oxirredução , Espectroscopia de Mossbauer/métodosRESUMO
Mössbauer spectra of the oxidized [Fe4S4]3+ and the reduced [Fe4S4]2+ clusters in the high-potential iron protein I from Ectothiorhodospira halophila were measured in a temperature range from 5 K to 240 K. EPR measurements and 57Fe electron-nuclear double resonance (ENDOR) experiments were carried out with the oxidized protein. In the oxidized state the cluster has a net spin S = 1/2 and is paramagnetic. As common in [Fe4S4]3+ clusters, the Mössbauer spectrum was simulated with two species contributing equally to the absorption area: two Fe3+ atoms couple to the "ferric-ferric" pair, and one Fe2+ and one Fe3+ atom give the "ferric-ferrous pair". For the simulation of the Mössbauer spectrum, g-values were taken from EPR measurements. A-tensor components were determined by 57Fe ENDOR experiments that turned out to be a necessary source of estimating parameters independently. In order to obtain a detailed agreement of Mössbauer and ENDOR data, electronic relaxation has to be taken into account. Relaxing the symmetry condition in a way that the electric field gradient tensor does not coincide with g- and A-tensors yielded an even better agreement of experimental and theoretical Mössbauer spectra. Spin-spin and spinlattice relaxation times were estimated by pulsed EPR; the former turned out to be the dominating mechanism at T = 5 K. Relaxation times measured by pulsed EPR and obtained from the Mössbauer fit were compared and yield nearly identical values. The reduced cluster has one additional electron and has a diamagnetic (S = 0) ground state. All the four irons are indistinguishable in the Mössbauer spectrum, indicating a mixed-valence state of Fe2.5+ for each.
Assuntos
Proteínas de Bactérias/química , Halorhodospira halophila/química , Proteínas Ferro-Enxofre/química , Complexo de Proteínas do Centro de Reação Fotossintética , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Conformação Proteica , Proteínas Recombinantes/química , Espectroscopia de MossbauerRESUMO
Protein dynamics can be characterized by the mean square displacements of the individual atoms of a molecule. This concept is extended to X-ray absorption spectroscopy (XAS) of proteins where the physical information in the Debye-Waller factor is in general neglected. In a first step, a procedure for the investigation of the temperature dependence of XAS spectra has been developed for a small iron compound. Subsequently, experiments have been performed on met-myoglobin. It is shown that the mean square displacements of XAS are smaller than those obtained by Mössbauer spectroscopy and far smaller than crystallographic mean square displacements. This behavior is explained by the different sensitivity of the methods. XAS measures a relative mean square displacement between the absorbing and backscattering atoms only. A comparison with mean square displacements calculated from normal modes shows that static displacements contribute significantly. It becomes obvious that the atoms of the active center show a high correlation of their motions.
Assuntos
Ferro , Metamioglobina/química , Espectrometria por Raios X/métodos , Sítios de Ligação , Biofísica/métodos , Modelos Químicos , Modelos Estatísticos , Modelos Teóricos , Conformação Proteica , Espalhamento de Radiação , Temperatura , Raios XRESUMO
Nuclear forward scattering of synchrotron radiation is used to determine the quadrupole splitting and the mean square displacement of the iron atom in deoxymyoglobin in the temperature range between 50 K and 243 K. Above 200 K an abnormally fast decay of the forward scattered intensity at short times after the synchrotron flash is observed, which is caused by protein-specific motions. The results strongly support the picture that protein dynamics seen at the position of the iron can be understood by harmonic motions in the low temperature regime while in the physiological regime diffusive motions in limited space are present. The shape of the resonance broadening function is investigated. An inhomogeneous broadening with a Lorentzian distribution indicating dipole interactions results in a better agreement with the experimental data than the common Gaussian distribution.
Assuntos
Mioglobina/análogos & derivados , Animais , Heme/química , Ferro , Cinética , Mioglobina/química , Mioglobina/efeitos da radiação , Espalhamento de Radiação , Espectroscopia de Mossbauer , Síncrotrons , Termodinâmica , BaleiasRESUMO
A metastable state of myoglobin is produced by reduction of metmyoglobin at low temperatures. This is done either by irradiation with x-rays at 80 K or by electron transfer from photoexcited tris(2, 2'-bipyridine)-ruthenium(II) at 20 K. At temperatures above 150 K, the conformational transition toward the equilibrium deoxymyoglobin is observed. X-ray crystallography, Raman spectroscopy, and temperature-dependent optical absorption spectroscopy show that the metastable state has a six-ligated iron low-spin center. The x-ray structure at 115K proves the similarity of the metastable state with metmyoglobin. The Raman spectra yield the high-frequency vibronic modes and give additional information about the distortion of the heme. Analysis of the temperature dependence of the line shape of the Soret band reveals that a relaxation within the metastable state starts at approximately 120 K. Parameters representative of static properties of the intermediate state are close to those of CO-ligated myoglobin, while parameters representative of dynamics are close to deoxymyoglobin. Thus within the metastable state the relaxation to the equilibrium is initiated by changes in the dynamic properties of the active site.