Versatile magnetometer assembly for characterizing magnetic properties of nanoparticles.

We constructed a versatile magnetometer assembly for characterizing iron oxide nanoparticles. The magnetometer can be operated at room temperature or inside a cryocooler at temperatures as low as 6 K. The magnetometer's sensor can be easily exchanged and different detection electronics can be used. We tested the assembly with a non-cryogenic commercial Hall sensor and a benchtop multimeter in a four-wire resistance measurement scheme. A magnetic moment sensitivity of 8.5 × 10(-8) Am(2) was obtained with this configuration. To illustrate the capability of the assembly, we synthesized iron oxide nanoparticles coated with different amounts of a triblock copolymer, Pluronic F-127, and characterized their magnetic properties. We determined that the polymer coating does not affect the magnetization of the particles at room temperature and demonstrates that it is possible to estimate the average size of coating layers from measurements of the magnetic field of the sample.

Dynamics of heme complexed with human serum albumin: a theoretical approach.

Human serum albumin (HSA) is the most abundant protein in the blood serum. It binds several ligands and has an especially strong affinity for heme, hence becoming a natural candidate for oxygen transport. In order to analyze the interaction of HSA-heme, molecular dynamics simulations of HSA with bound heme were performed. Based on the results of X-ray diffraction, the binding site of the heme, localized in subdomain IB, was considered. We analyzed the fluctuations and their correlations along trajectories to detect collective motions. The role of H bonds and salt bridges in the stabilization of heme in its pocket was also investigated. Complementarily, the localization of water molecules in the hydrophobic pocket and the interaction with heme were discussed.

Hydration of hydrophobic biological porphyrins.

Explicit solvent, single solute molecular dynamics simulations of protoporphyrin IX and its Fe(2+) complex (heme) in water were performed. The relation of solute-solvent was examined through the spatial distribution functions of water molecules around the centroid of the porphyrin ring. A detailed description of the time-averaged structure of water surrounding the solutes as well as of its fluctuations is presented.

Quenching of the intrinsic fluorescence of bovine serum albumin by chlorpromazine and hemin.

The binding of chlorpromazine (CPZ) and hemin to bovine serum albumin was studied by the fluorescence quenching technique. CPZ is a widely used anti-psychotic drug that interacts with blood components, influences bioavailability, and affects function of several biomolecules. Hemin is an important ferric residue of hemoglobin that binds within the hydrophobic region of albumin with high specificity. Quenching of the intrinsic fluorescence of bovine serum albumin (BSA) was observed by selectively exciting tryptophan residues at 290 nm. Emission spectra were recorded in the range from 300 to 450 nm for each quencher addition. Stern-Volmer graphs were plotted, and the quenching constant estimated for BSA solution titrated with hemin at 25 masculine C was 1.44 (+/- 0.05) x 10(5) M(-1). Results showed that bovine albumin tryptophans are not equally accessible to CPZ, in agreement with the idea that polar or charged quenchers have more affinity for amino acid residues on the outer wall of the protein. Hemin added to albumin solution at a molar ratio of 1:1 quenched about 25% of their fluorescence. The quenching effect of CPZ on albumin-hemin solution was stronger than on pure BSA. This increase can be the result of combined conformational changes in the structure of albumin caused firstly by hemin and then by CPZ. Our results suggest that the primary binding site for hemin on bovine albumin may be located asymmetrically between the two tryptophans along the sequence formed by subdomains IB and IIA, closer to tryptophan residue 212.

Quenching of the intrinsic fluorescence of bovine serum albumin by chlorpromazine and hemin

The binding of chlorpromazine (CPZ) and hemin to bovine serum albumin was studied by the fluorescence quenching technique. CPZ is a widely used anti-psychotic drug that interacts with blood components, influences bioavailability, and affects function of several biomolecules. Hemin is an important ferric residue of hemoglobin that binds within the hydrophobic region of albumin with high specificity. Quenching of the intrinsic fluorescence of bovine serum albumin (BSA) was observed by selectively exciting tryptophan residues at 290 nm. Emission spectra were recorded in the range from 300 to 450 nm for each quencher addition. Stern-Volmer graphs were plotted, and the quenching constant estimated for BSA solution titrated with hemin at 25ºC was 1.44 (± 0.05) x 10(5) M-1. Results showed that bovine albumin tryptophans are not equally accessible to CPZ, in agreement with the idea that polar or charged quenchers have more affinity for amino acid residues on the outer wall of the protein. Hemin added to albumin solution at a molar ratio of 1:1 quenched about 25 percent of their fluorescence. The quenching effect of CPZ on albumin-hemin solution was stronger than on pure BSA. This increase can be the result of combined conformational changes in the structure of albumin caused firstly by hemin and then by CPZ. Our results suggest that the primary binding site for hemin on bovine albumin may be located asymmetrically between the two tryptophans along the sequence formed by subdomains IB and IIA, closer to tryptophan residue 212.

Inhibition of acetylcholinesterase from Electrophorus electricus (L.) by tricyclic antidepressants.

The effects of tricyclic antidepressants drugs (TCA) amitriptyline, imipramine and nortriptyline, on purified Electrophorus electricus (L.) acetylcholinesterase (AChE; acetylcholine hydrolase, EC 3.1.1.7) were studied using kinetic methods and specific fluorescent probe propidium. The antidepressants inhibited AChE activity by a non-competitive mechanism. Inhibition constants range from 200 to 400 microM. Dimethylated amitriptyline and imipramine were more potent inhibitors than the monomethylated nortriptyline. Fluorescence measurements using bis-quaternary ligand propidium were used to monitor ligand-binding properties of these cationic antidepressants to the AChE peripheral anionic site (PAS). This ligand exhibited an eight-fold fluorescence enhancement upon binding to the peripheral anionic site of AChE from E. electricus (L.) with K(D)=7 x 10(-7)M. It was observed that TCA drugs displaced propidium from the enzyme. On the basis of the displacement experiments antidepressant dissociation constants were determined. Similar values for the inhibition constants suggest that these drugs have similar affinity to the peripheral anionic site. The results also indicate that the catalytic active center of AChE does not participate in the interaction of enzyme with tricyclic antidepressants. These studies suggest that the binding site for tricyclic antidepressants is located at the peripheral anionic site of E. electricus (L.) acetylcholinesterase.

Denervation alters protein-lipid interactions in membrane fractions from electrocytes of Electrophorus electricus (L.).

Protein-lipid interactions are studied in normal and denervated electrocytes from Electrophorus electricus (L.). Structural modifications of the lipid micro-environment encircling integral membrane proteins in membrane fractions presenting Na(+),K(+)-ATPase activity are investigated using ESR spectroscopy of stearic acid spin labeled at the 14th carbon (14-SASL). The microsomal fraction derived from the innervated electric organ exhibits, on a discontinuous sucrose gradient, a bimodal distribution of the Na(+),K(+)-ATPase activity, bands a and b. Band b is almost absent in microsomes from the denervated organ, and band a', with the same density as band a has lower Na(+),K(+)-ATPase activity. Band a' presents a larger ratio of protein-interacting lipids than band a. Analysis of the lipid stoichiometry at the protein interface indicates that denervation causes at least a twofold average decrease on protein oligomerization. Physical inactivity and denervation have similar effects on protein-lipid interactions. Denervation also influences the selectivity of proteins for fatty acids. Experiments in decreasing pH conditions performed to verify the influence of stearic acid negative charge on protein interaction revealed that denervation produces loss of charge selectivity. The observed modifications on molecular interactions induced by denervation may have importance to explain modulation of enzyme activity.

Carboxyl groups at the membrane interface as molecular targets for local anesthetics.

The interaction of the tertiary amine drugs chlorpromazine and dibucaine in their cationic form with carboxyl groups at the membrane surface is studied at concentrations relevant to anesthesia. Spin-labeled stearic acid is used both to provide the carboxyl groups and to monitor binding and ionization behavior in egg lecithin liposomes. Membrane anesthetic concentrations are spectrophotometrically obtained. They are shown to determine the drug influence on carboxyl groups at the membrane surface, independently of aqueous concentrations. The intramembrane association constants (related to the usual aqueous phase ones through the partition coefficient) of the drugs with fatty acids are determined. The same value (10(2) M-1) is obtained for both drugs, suggesting that it is approximately the same for all tertiary amine local anesthetics. pH titrations of anesthetic-treated spin-labeled membranes are performed. The observed shifts in the fatty acid pK are higher than can be produced assuming uniform distribution of the drug in the membrane surface, implying that there is an increased affinity of local anesthetics for superficial carboxyl. This affinity could account for the resting block of voltage-gated Na+ channels. Under these considerations, local anesthetic binding sites at voltage-gated Na+ channels and at sarcoplasmic reticulum Ca(2+)-ATPase are proposed.

Binding and location of dipyridamole derivatives in micelles: the role of drug molecular structure and charge.

Binding and localization of the vasodilator and antitumor drug coactivator dipyridamole (DIP) and of its three derivatives, RA14, RA47 and RA25 (DIPD), to cationic (cetyltrimethylammonium chloride), anionic (sodium dodecylsulfate), zwitterionic (N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate), and neutral (t-octylphenoxypolyethoxyethanol) micelles was studied using fluorescence, optical absorption and 1H NMR spectroscopy. The analysis of NMR, optical absorption and fluorescence data indicates that the depth of localization of the drugs in the micelles from the surface decreased in the order DIP > RA14 > RA47 > RA25. The binding constants for the neutral drug forms change in the same order in the range of 1400-3100 M-1 for DIP to 80-300 M-1 for RA25. This order is identical with the reported biological activity of DIPD. For the protonated drugs in zwitterionic or neutral micelles the binding constants are reduced by a factor of 20-75.

Interaction of alkanols and local anesthetics with spin-labeled Ca(2+)-ATPase of sarcoplasmic reticulum vesicles.

Alkanols and tertiary amine derivative local anesthetics modify the activity of Ca(2+)-ATPase. In order to investigate the primary binding sites, associated to the functional changes, sarcoplasmic reticulum (SR) Ca(2+)-ATPase was labeled with maleimide derivative spin labels which bind covalently to SH groups of cysteine residues and allow to probe the regions of the protein close to those residues. The EPR measurements showed motional constraints induced by drug-treatment which indicate changes in the enzyme dynamics and structure. n-Alkanols are shown to affect some of the protein-bound labels by restricting their motion. There is, however, no correlation between the functional effects and the observed motional restriction, in the sense that concentrations of the different alcohols leading to the same functional effects do not induce the same degree of restriction. Dibucaine and tetracaine at functional relevant concentrations also restrict the movement of protein bound labels. But, in this case, correlation between spectral changes and functional effects is observed.

Charge- and pH-dependent binding sites for dibucaine in ionic micelles: a fluorescence study.

Binding of micromolar concentrations of the local anesthetic dibucaine to micelles of cationic, zwitterionic and anionic detergents was studied using the fluorescence emission of dibucaine. Difference in quantum yields for charged and neutral dibucaine allowed to obtain shifts of pKa values due to binding. Estimates for the electrostatic potential affecting the tertiary amine of dibucaine were obtained from the pKa shifts. Change of fluorescence emission upon binding allowed to obtain the binding constants of both charged and neutral dibucaine to the micelles. The binding constant for the neutral form is essentially independent of micelle charge and of specific differences in detergent structure. Consistency between the ratio of neutral to cationic dibucaine binding constants and the measured pKa shift was tested. For LPC micelles complete agreement was found. For CTAC, however, the ratio of binding constants does not explain the pKa shift. The discrepancy between the results is used to estimate the errors involved upon neglecting non-coulombic electrostatic interactions of drugs to charged membrane surfaces. Fluorescence quenching with sodium iodide and nitroxide stearic acid derivatives allowed a depth profiling of the drug in the micelles.

Depth profiling of dibucaine in sarcoplasmic reticulum vesicles by fluorescence quenching.

The location of molecules of the local anesthetic dibucaine in sarcoplasmic reticulum vesicles (SRV) was determined using the quenching of its intrinsic fluorescence by iodide and by nitroxide-labeled stearic acids (SASL) with the nitroxide group at different positions of the fatty acyl chain. The molar ratios of dibucaine to Ca(2+)-ATPase in the samples were less than 1. The acid-base titration of membrane bound dibucaine revealed a pK of 9.1, showing a negligible shift upon binding. The quenching data were obtained at pH 6.8 and are therefore related to protonated dibucaine. Quenching by iodide showed SRV-bound dibucaine to be more protected from collisions with iodide anion than dibucaine in buffer or even in neutral micelles. This shows the influence of negatively charged lipids in keeping iodide away from the ionic diffuse layer of the membrane surface where the dibucaine tertiary amine might be located. Analysis of the SASL quenching data indicates that dibucaine molecules are at a shallow position in the membrane bilayer. Their average depth was found to be at most that of the fourth carbon atom of the fatty acyl chain. The results do not exclude a preferential site for dibucaine in Ca(2+)-ATPase, but if there is such site it must be located at the protein/lipid interface.

The effects of n-alkanols on the lipid/protein interface of Ca(2+)-ATPase of sarcoplasmic reticulum vesicles.

The effects of ethanol, n-butanol, n-hexanol and n-octanol on lipid-protein interactions in sarcoplasmic reticulum vesicles (SRV) are investigated using the C-14 nitroxide spin-labeled phosphatidylcholine. n-Alkanols, which activate the Ca(2+)-dependent ATPase of sarcoplasmic reticulum but decrease net Ca2+ uptake by the vesicles, are shown to affect the lipids interacting with the protein surface. Spectral analysis revealed that increasing concentrations of the alcohols progressively displace and mobilize lipids from the lipid/protein interface. For butanol, hexanol and octanol maximally activated SRV, 23 to 30% of the protein-interacting lipids are displaced. Thus, the displacement of more than 30% of the annular lipids by these alkanols cause inhibition of the enzyme. The motional properties of the labels that remain restricted by the protein surface are unaffected by the alcohols. The degree of mobilization attained by the labels displaced from the interface is much greater than that observed in alcohol-treated dispersions of extracted lipids. We propose that the alcohol molecules interfere with the protein-lipid interactions creating fluid clusters around the proteins. These fluidized regions would affect the enzyme conformation, perturbing its function. Fluidized annular lipids apparently increase the number of ion-conducting defects around the enzyme, increasing Ca2+ efflux, and thereby reducing net uptake.

A study of the effect of general anesthetics on lipid-protein interactions in acetylcholine receptor enriched membranes from Torpedo nobiliana using nitroxide spin-labels.

Stearic acid, phosphatidylcholine, and phosphatidylglycerol nitroxide spin-labels were used to probe the effect of 1-hexanol, urethane, diethyl ether, and ethanol on lipid-protein interactions in nicotinic acetylcholine receptor (nAcChoR) rich membranes from Torpedo nobiliana. For stearic acid spin-labeled at the C-14 position of the sn-1 acyl chain, 1-hexanol induced little change (over a wide concentration range, 0-16.7 mM) in either the ESR line shape or the proportion of motionally restricted spectral component from labels probing the protein interface. The main effect of 1-hexanol was limited to an increase in the mobility of stearic acid spin-labels probing the non-protein-associated environment. In contrast, for C-14 phosphatidylcholine spin-label, 1-hexanol decreased the fraction of spin-labels motionally restricted at the protein interface from 0.33 without 1-hexanol to 0.20 with 16.7 mM 1-hexanol, with no change in the line shape of the spectral component of these labels. The ESR spectral line shape of the fluid component due to phosphatidylcholine labels in sites away from the protein interface displayed a gradual decrease in spectral anisotropy on addition of increasing amounts of 1-hexanol. At a concentration of 1-hexanol that desensitizes half the receptors, the fraction of motionally restricted phosphatidylcholine spin-label is reduced by approximately 15%. The effect of 1-hexanol on phosphatidylglycerol spin-labels was intermediate between these two cases. Similar effects were measured with other general anesthetics, including urethane, diethyl ether, and ethanol.(ABSTRACT TRUNCATED AT 250 WORDS)

pH-dependent phase transition of chlorpromazine micellar solutions in the physiological range.

The effects of pH and drug concentration on aggregation properties of chlorpromazine-HCl (CPZ) are examined. The critical micelle concentration (cmc) changes from 0.2 mM at pH 7.3 to 2 mM at pH 5.6 as estimated from the stearic acid spin label solubility measurements. For concentrations above the cmc CPZ micelles undergo a concentration-, temperature- and pH-dependent transition leading to phase separation. This phase transition is followed by a sudden increase of light scattering. The phase diagram pH vs. concentration is obtained by observation of the cloud point for concentrations ranging from 0.01 to 10 mM. The intramicellar environment is probed at pH ranging from 5.5 to 8.0 using a stearic acid spin label. The intramicellar compactness increases smoothly with increasing pH suggesting the weakening of polar heads repulsion due to charge decrease. The reported results indicate that pH effects are relevant and should be properly taken into account in the performance and interpretation of experiments with CPZ.

Magnetic localisation of a current dipole implanted in dogs.

In order to evaluate the difficulty in localising a current dipole due to volume conductor current contributions to the magnetocardiogram, accuracy of depth localisation of a commercial coaxial pacemaker cable, used as a current dipole, was studied in two experimental situations: immersed in a prismatic container with NaCl solution and introduced into the lower oesophagus of dogs. Isofield contour maps were obtained by interpolation of the magnetic field measured over a plane and perpendicular to it with a third-order gradiometer coupled to a SQUID. The dipole can be accurately localised in the prismatic container. The observation of an isofield map that is symmetric about the maximum-minimum axis when the dogs are in the dorsal decubitus position with the dipole in the cephalocaudal direction implies that internal inhomogeneities in the dog's volume conductor produce no appreciable effect on the magnetic field. Nevertheless, a large distortion of the magnetic field lines is observed and can be explained by calculations using models that take into account the external boundary of the volume conductor.

EPR spectral changes of nitrosyl hemes and their relation to the hemoglobin T-R transition.

EPR spectra of nitrosyl hemes were used to study the quaternary structure of hemoglobin. Human adult hemoglobin has been titrated with nitric oxide at pH 7.0 and 25 degrees C. After the equilibration of NO among the alpha and beta subunits the samples were frozen for EPR measurements. The spectra were fitted by linear combinations of three standard signals: the first arising from NO-beta-hemes and the other two arising for NO-alpha-hemes of molecules in the high- and low-affinity conformations. The fractional amounts of alpha subunits exhibiting the high-affinity spectrum fitted the two-state model (Edelstein, S.J. (1974) Biochemistry 13, 4998-5002) with the allosteric constant L = 7.10(6) and relative affinities cNO alpha and cNO beta approx. 0.01. Hemoglobin has been marked with nitric oxide one chain using low-saturation amounts of nitric oxide. The EPR spectra was studied as a function of oxygen saturation. Linear combinations of the three standard signals above fitted these spectra. The fractions of molecules exhibiting the high-affinity spectrum fitted the two-state model with L = 7 . 10(6), c)2 = 0.0033 and cNO alpha = 0.08, instead of cNO alpha = 0.01. Thus, the two-state model is not adequate to describe the conformational transition of these hybrids. The results present evidence of the non-equivalence between oxygen and nitric oxide as ligands.