ABSTRACT
Synthesis, spectral properties, and photodynamic activity of water-soluble amino acid fullerene C60 derivatives (AFD) and four original AFD-PPa dyads, obtained by covalent addition of dye pyropheophorbide (PPa) to AFD, were studied. In aqueous solution, these AFD-PPa dyads form nanoassociates as a result of self-assembly. In this case, a significant change in the absorption spectra and strong quenching of the dye fluorescence in the structure of the dyads were observed. A comparison of superoxide or singlet oxygen generation efficiency of the studied compounds in an aqueous solution showed the photodynamic mechanism switching from type II (singlet oxygen generation of the native dye) to I type (superoxide generation of dyads). All dyads have pronounced phototoxicity on cells Hela with IC50 9.2 µM, 9.2 µM, 12.2 µM for dyads Val-C60-PPa, Ala-C60-PPa and Pro-C60-PPa, respectively. Such facilitation of type I photodynamic mechanism could be perspective against hypoxic tumors.
ABSTRACT
The effects of the newly synthesized covalent conjugates of water-soluble fullerene derivatives (WSFD) with xanthene dyes: polyanionic WSFD-fluorescein (1), polycationic WSFD-fluorescein (2), polyanionic WSFD-eosin (3), and polyanionic WSFD (4), polycationic WSFD (5), fluorescein (6) and eosin (7), on activity of the membrane-bound Ca2+-ATPase of the sarcoplasmic reticulum (SR Ca2+-ATPase) were studied. Compounds 1, 3, 4, 6, and 7 inhibit the hydrolytic function of the enzyme, the inhibition constants for these compounds are Ki=1.3×10-5 M (1), Ki=4.7×10-6 M (3), Ki=2.5×10-6 M (4), Ki=6.1×10-5 M (6), and Ki=5.8×10-6 M (7). The effects of compounds 3, 6, and 7 on the hydrolytic function of the enzyme is competitive; compounds 1 and 4 are noncompetitive. Polycationic WSFD fluorescein (2) and polycationic WSFD (5) do not affect ATP hydrolysis, but inhibit active Ca2+ transport in a concentration of 0.01 mM by 100±10 and 40±4%, respectively. Conjugates 1 and 3 completely inhibit the hydrolytic and transport functions of the enzyme in a concentration of 0.01 mM, and in a concentration of 0.001 mM inhibit active Ca2+ transport by 60±6 and 55±6% uncoupling the hydrolytic and transport functions of SR Ca2+-ATPases. The obtained results demonstrate a significant effect of the studied compounds on the active transmembrane transfer of Ca2+ and make it possible to predict the presence of antimetastatic and antiaggregatory activities of the studied compounds.
Subject(s)
Calcium-Transporting ATPases/drug effects , Fullerenes/pharmacology , Sarcoplasmic Reticulum/enzymology , Xanthenes/pharmacology , Animals , Calcium/metabolism , Calcium-Transporting ATPases/antagonists & inhibitors , Calcium-Transporting ATPases/metabolism , Coloring Agents/chemistry , Coloring Agents/pharmacology , Fullerenes/chemistry , Humans , Kinetics , Protein Binding/drug effects , Sarcoplasmic Reticulum/drug effects , Sarcoplasmic Reticulum/metabolism , Xanthenes/chemistryABSTRACT
The effect of N-nitroxymethyl succinimide (1), N-(2-nitroxyethyl) succinimide (2) and N-(3-nitroxypropyl) succinimide (3) on enzymatic activity of cyclic guanosine monophosphate (cGMP) phosphodiesterase was studied and crystal structure of compound (2) was determined. It was shown that all studied N-nitroxy succinimides inhibited cGMP phosphodiesterase in a concentration range of 0.1-0.001 mM. Compound (2) noncompetitively and reversibly inhibited hydrolytic function of enzyme with Ki=1.7×10-5 Ð. Inhibition constant for the reference compound N-(2-nitroethyl) nicotinamide (nicorandil) was 3×10-5 Ð.
Subject(s)
Cyclic GMP/metabolism , Guanosine Monophosphate/metabolism , Phosphoric Diester Hydrolases/metabolism , Succinimides/pharmacology , Animals , Enzyme Activation/drug effects , Kinetics , Rats , Rats, WistarABSTRACT
The effect of iron nitrosyl complexes, NO donors, of a general formula [Fe2(L)2(NO)4] with functional sulfur-containing ligands (L-3-nitro-phenol-2-yl, 4-nitro-phenol-2-yl, or 1-methyl-tetrazol-5-yl) on the activity of sarcoplasmic reticulum Ca2+-ATPase and cyclic guanosine monophosphate phosphodiesterase (cGMP PDE) was studied. The test complexes uncoupled the hydrolytic and transport functions of Ca2+- ATPase, thus disturbing the balance of Ca2+ ions in cells, which may affect the formation of thrombi and adhesion of metastatic cells to the endothelium of capillaries. They also inhibited the activity of cGMP PDE, thereby contributing to the accumulation of the second messenger cGMP. The studied iron nitrosyl complexes can be considered as potential drugs.
Subject(s)
Calcium-Transporting ATPases/metabolism , Cyclic GMP/metabolism , Iron/pharmacology , Nitric Oxide Donors/pharmacology , Nitrogen Oxides/pharmacology , Phosphoric Diester Hydrolases/metabolism , Sarcoplasmic Reticulum/enzymology , Animals , Humans , Hydrolysis/drug effectsABSTRACT
In the cycle of photosynthetic reaction centers, the initially oxidized special pair of bacteriochlorophyll molecules is subsequently reduced by an electron transferred over a chain of four hemes of the complex. Here, we examine the kinetics of electron transfer between the proximal heme c-559 of the chain and the oxidized special pair in the reaction center from Rps. sulfoviridis in the range of temperatures from 294 to 40 K. The experimental data were obtained for three redox states of the reaction center, in which one, two, or three nearest hemes of the chain are reduced prior to special pair oxidation. The experimental kinetic data are analyzed in terms of a Sumi-Marcus-type model developed in our previous paper,1 in which similar measurements were reported on the reaction centers from Rps. viridis. The model allows us to establish a connection between the observed nonexponential electron-transfer kinetics and the local structural relaxation dynamics of the reaction center protein on the microsecond time scale. The activation energy for relaxation dynamics of the protein medium has been found to be around 0.1 eV for all three redox states, which is in contrast to a value around 0.4-0.6 eV in Rps. viridis.1 The possible nature of the difference between the reaction centers from Rps. viridis and Rps. sulfoviridis, which are believed to be very similar, is discussed. The role of the protein glass transition at low temperatures and that of internal water molecules in the process are analyzed.
Subject(s)
Electrons , Photosynthetic Reaction Center Complex Proteins/chemistry , Photosynthetic Reaction Center Complex Proteins/metabolism , Rhodopseudomonas/enzymology , Kinetics , Models, Molecular , Protein Structure, TertiaryABSTRACT
In a recent work from this group (Popovic, D. M.; Stuchebrukhov A. A. FEBS Lett. 2004, 566, 126), a model of proton pumping by cytochrome c oxidase (CcO) was proposed. The key element of the model is His291 (bovine notation), a histidine ligand to enzyme's CuB redox center, which plays the role of the pump element. The model assumes that upon electron transfer between heme a and the binuclear catalytic center of the enzyme, two sequential proton transfers occur: First, a proton from Glu242 is transferred to an unprotonated His291, then a second proton, after reprotonation of Glu242 from the negative side of the membrane, is transferred to a hydroxyl group in the binuclear center, a water molecule is formed, and the first proton, due to proton-proton repulsion, is expelled from His291 to the positive side of the membrane, resulting in a pumping event. In the process the free energy of water formation (i.e., reduction of oxygen) is transformed into a proton gradient across the membrane. The model possesses specific kinetic features. It assumes, for example, that upon electron transfer the first proton is transferred to the proton-loading site of the pump, His291, and not to the catalytic center of the enzyme. Here, we analyze the kinetic properties of the proposed model, and calculate the time dependence of the membrane potential generated by CcO upon a single electron injection into the enzyme. These data are directly compared with recent experimental measurements of the membrane potential generated by CcO. Specifically, F to O, and O to E transitions will be discussed. Several enzymes from different organisms (bovine, two bacterial enzymes, and several mutants) are compared and discussed in detail. The kinetic description, however, is phenomenological, and does not include explicitly the nature of the groups involved in proton translocation, except in terms of their position depth within the membrane; thus, the kinetic equations developed here are in fact describe a generic model, similar, e.g., to that proposed earlier by Peter Rich (P.R. Rich, Towards an understanding of the chemistry of oxygen reduction and proton translocation in the iron-copper respiratory oxidases. Aust. J. Plant Physiol. 22 (1995) 479-486), and which is based on the idea of displacement of the pumped protons by the chemical ones.
Subject(s)
Electron Transport Complex IV/metabolism , Animals , Cattle , Electron Transport , Electron Transport Complex IV/genetics , Kinetics , Membrane Potentials , Models, Biological , Mutation/genetics , Paracoccus denitrificans/enzymology , Paracoccus denitrificans/metabolism , Protons , Rhodobacter sphaeroides/enzymology , Rhodobacter sphaeroides/metabolismABSTRACT
The broad set of nonexponential electron transfer (ET) kinetics in reaction centers (RC) from Rhodopseudomonas sulfoviridis in temperature range 297-40 K are described within a mixed adiabatic/nonadiabatic model. The key point of the model is the combination of Sumi-Marcus and Rips-Jortner approaches which can be represented by the separate contributions of temperature-independent vibrational (v) and temperature-dependent diffusive (d) coordinates to the preexponential factor, to the free energy of reaction DeltaG=DeltaG(v)+DeltaG(d)(T) and to the reorganization energy lambda=lambda(v)+lambda(d)(T). The broad distribution of protein dielectric relaxation times along the diffusive coordinate is considered within the Davidson-Cole formalism.
Subject(s)
Bacterial Proteins/chemistry , Bacteriochlorophylls/chemistry , Cytochromes/chemistry , Models, Chemical , Rhodopseudomonas/chemistry , Dimerization , Kinetics , ThermodynamicsABSTRACT
A photochromic compound, stilbazolium betaine M, when associated with metmyoglobin undergoes an accelerated thermal cis-trans isomerization. A study of the pH and ionic strength dependence of the isomerization reaction rate of the photochrome associated with metmyoglobin was performed. A comparative investigation of the reaction carried out in the presence of three proteins, metmyoglobin, apomyoglobin, and human albumin, indicates a specific influence of the heme pocket environment on the reaction. Possible mechanisms of the reaction acceleration are considered.
Subject(s)
Benzylidene Compounds/metabolism , Dihydropyridines/metabolism , Metmyoglobin/metabolism , Albumins/metabolism , Apoproteins/metabolism , Benzylidene Compounds/chemistry , Catalysis , Dihydropyridines/chemistry , Heme/chemistry , Humans , Hydrogen-Ion Concentration , Kinetics , Models, Structural , Myoglobin/metabolism , Osmolar Concentration , Stereoisomerism , TemperatureABSTRACT
The modifying effects of the products of the equimolar addition Of DL-alanine and DL-alanyl-DL-alanine to fullerene C60 on the structure and permeability of the lipid bilayer of phosphatidylcholine liposomes has been studied using the luminescence probe technique. It is shown that these water soluble amino acid and dipeptide derivatives of fullerene (C60-AD) are quenchers of pyrene fluorescence and erythrosine phosphorescence of in both a water solution and liposomes. To study the permeability of the lipid bilayer a procedure based on the triplet probe technique has been developed. It has been found that the C60-AD derivatives under study are able to localize inside the artificial membrane, to penetrate into the liposomes through the lipid bilayer and to perform activated transmembrane transport of bivalent metal ions.
Subject(s)
Carbon/chemistry , Carbon/pharmacology , Fullerenes , Lipid Bilayers/chemistry , Liposomes/chemistry , Alanine/chemistry , Biological Transport , Cobalt/chemistry , Cobalt/metabolism , Erythrosine/chemistry , Kinetics , Peptides/chemistry , Permeability , Phosphatidylcholines/chemistry , Pyrenes/chemistry , Solubility , Spectrometry, Fluorescence/methods , Titrimetry , WaterABSTRACT
The physical principles of several new approaches to the investigation of biological and model systems are discussed, including versions of the spin label method based on relaxation measurements, and also the methods of triplet, Mössbauer, electron-scattering and radical-pair labels and probes. It is shown that all these methods make it possible to investigate molecular mobility of the medium with the correlation frequencies tau c-1 = 10(-3) -10(11) s-1, to measure the rate constants of collisions Ktr = 10(3) -10(10) M-1 s-1, to measure the distance between centers up to 100 A and finally, to evaluate the immersion depths of paramagnetic and chromophore centers in matrices up to 40 A. The combined approach is demonstrated with examples from studies of the structure of nitrogenase, the reaction centers of photosynthetic bacteria and sarcoplasmic reticulum membranes and from studies of the molecular dynamics of proteins and membranes.