Water translational motion at the bilayer interface: an NMR relaxation dispersion measurement.

Nuclear magnetic relaxation rates for water protons in aqueous palmitoyloleoylphosphatidylcholine vesicle suspensions containing different nitroxide free radical spin labels are reported as a function of magnetic field strength corresponding to proton Larmor frequencies from 10 kHz to 30 MHz. Under these conditions the water proton relaxation rate is determined by the magnetic coupling between the water protons and the paramagnetic nitroxide fixed on the phospholipid. This coupling is made time-dependent by the relative translational motion of the water proton spins past the nitroxide radical. Using theories developed by Freed and others, we interpret the NMR relaxation data in terms of localized water translational motion and find that the translational diffusion constant for water within approximately 10 A of the phospholipid surface is 6 x 10(-10) m2 s(-1) at 298 K. Similar results are obtained for three different nitroxide labels positioned at different points on the lipid. The diffusion is a thermally activated process with an activation energy only slightly higher than that for bulk water.

Simulation of saturation transfer electron paramagnetic resonance spectra for rotational motion with restricted angular amplitude.

We have simulated both conventional (V1) and saturation transfer (V'2) electron paramagnetic resonance spectra for the case of Brownian rotational diffusion restricted in angular amplitude. Numerical solutions of the diffusion-coupled Bloch equations were obtained for an axially symmetric 14N nitroxide spin label with its principal axis rotating within a Gaussian angular distribution of full width delta theta at half maximum. Spectra were first calculated for a macroscopically oriented system with cylindrical symmetry (e.g., a bundle of muscle fibers or a stack of membrane bilayers), with the Gaussian angular distribution centered at theta 0 with respect to the magnetic field. These spectra were then summed over theta 0 to obtain the spectrum of a randomly oriented sample (e.g., a dispersion of myofibrils or membrane vesicles). The angular amplitude delta theta was varied from 0 degrees, corresponding to isotropic motion (order parameter = 0). For each value of delta theta, the rotational correlation time, tau r, was varied from 10(-7) to 10(-2) s, spanning the range from maximal to minimal saturation transfer. We provide plots that illustrate the dependence of spectral parameters on delta theta and tau r. For an oriented system, the effects of changing delta theta and tau r are easily distinguishable, and both parameters can be determined unambiguously by comparing simulated and experimental spectra. For a macroscopically disordered system, the simulated spectra are still quite sensitive to delta theta, but a decrease in tau r produces changes similar to those from an increase in delta theta. If delta theta can be determined independently, then the results of the present study can be used to determine tau r from experimental spectra. Similarly, if tau r is known, then delta theta can be determined.

Interaction between human serum albumin and alpha 1-acid glycoprotein in the binding of lidocaine to purified protein fractions and sera.

The authors have determined the binding parameters for [14C] lidocaine to several preparations of purified human serum alpha 1-acid glycoprotein (AAG) and human serum albumin (HSA). The authors find that the average Kd for the three different purified AAG preparations was 15.2 +/- 0.5 microM, with an average of 0.567 +/- 0.009 binding sites (N) per AAG molecule, when the concentration of AAG was determined by a standard colorimetric protein assay. The number of binding sites per AAG increased to 0.99 +/- 0.06 when the concentration of AAG was determined immunologically. On the other hand, delipidized AAG had a Kd of 28.0 microM, with an N of 1.72 based on the immunological assay. Various preparations of HSA had Kd/N values ranging from 4.3 to 17.3 mM. The number of binding sites per HSA molecule could not be determined because the maximum concentration of lidocaine that can be used is 10 mM, which is only approximately equal to the Kd. The Kd to HSA was so low and the capacity so high that, at therapeutic concentrations, the binding was in the linear range. Solutions containing both purified AAG and HSA had Kd values of 30.2 +/- 1.8 microM, with 2.01 +/- 0.04 binding sites per AAG molecule. With HSA and delipidized AAG, the Kd was 60 microM, with 3.16 sites per delipidized AAG molecule. This increase in the binding parameters appeared to be due to a direct interaction between the two proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

The magnetic field dependence of transverse water proton relaxation induced by nitroxide radicals.

The magnetic field dependence of the water proton T2 is calculated for aqueous solutions of nitroxides based on a detailed analysis of early T1 measurements on nitroxide solutions made as a function of magnetic field. The results parallel those for T1 closely and, unlike metal systems, the implications for magnetic imaging are similar.

No detectable reaction of the anion radical metabolite of nitrofurans with reduced glutathione or macro-molecules.

The initial metabolite formed by most mammalian nitroreductases is the nitro anion free radical. We, as well as others, have proposed that nitroheterocyclic anion radicals covalently bind to protein, DNA, or thiol compounds such as reduced glutathione (GSH). Our results indicate that even at 100 mM GSH does not affect the steady-state concentration of the nitro anion free radical of N-[4-(5-nitro-2-furyl)-2-thiazolyl]acetamide (NFTA) in rat hepatic microsomal or xanthine oxidase incubations. The steady-state ESR amplitude of the anion radical is also unchanged by the addition of BSA or DNA. Similar results are obtained with nitrofurazone and nitrofurantoin. The reactive chemical species which binds to tissue macromolecules and GSH upon the reduction of nitrofurans remains unknown, but the anion free radical metabolite can be excluded from consideration.

Cytochrome c/H2O2-mediated one electron oxidation of carcinogenic N-fluorenylacetohydroxamic acids to nitroxyl free radicals.

The oxidation of carcinogenic hydroxamic acids, N-hydroxy-N-2-fluorenylacetamide (N-OH-2-FAA) and N-hydroxy-N-3-fluorenylacetamide (N-OH-3-FAA) catalyzed by horseradish peroxidase (HRP) or cytochrome c in the presence of H2O2 was investigated. HRP/H2O2 was a more efficient system in oxidation of both hydroxamic acids and the standard substrate, guaiacol, then cytochrome c/H2O2. Peroxidative activity of cytochrome c was shown after incubation with Triton X-100 and H2O2 for 20 min at room temperature in 0.05 M phosphate buffer (pH 7.5) or in 0.1 M sodium acetate (pH 6.0) without Triton X-100. Both hydroxamic acids were oxidized to nitroxyl free radicals as shown by electron spin resonance (ESR) spectroscopy. These radicals dismutated to equimolar amounts of 2- or 3-nitrosofluorene and acetate esters of the corresponding hydroxamic acids as shown by thin layer chromatography and spectrophotometric analysis of the products. In addition, large amounts of the N-fluorenylamides were generated in the reactions with cytochrome c/H2O2 system. Of the products, only 2- or 3-nitrosofluorene per se or when generated from the oxidation of the hydroxamic acids, interacted with lecithin (1 mg/ml) to yield ESR signals of the immobilized nitroxyl free radicals. In contrast to HRP/H2O2 system, in which the initial velocity of the radical formation was too fast to measure and the maximal concentrations of the nitroxyl free radicals of both hydroxamic acids were similar, in the cytochrome c/H2O2 system the nitroxyl free radical of N-OH-2-FAA formed at a 6-fold faster rate and accumulated at a 2-fold higher concentration than the radical of N-OH-3-FAA. In both enzyme systems, the persistence of the signal and the length of time before it had decreased to one half its maximum were several-fold longer for the nitroxyl free radical of N-OH-3-FAA than for that of N-OH-2-FAA. These data showed that these nitroxyl free radicals differed in their kinetic properties. One electron oxidation of N-OH-3-FAA by HRP/H2O2 system and of both isomeric hydroxamic acids by cytochrome c/H2O2 system are reported for the first time in this work and may be considered an activation reaction in carcinogenesis by these compounds.

Spin-label and deuterium order parameter discrepancies in bilayers: one possible explanation.

We have simulated electron spin resonance spectra of anisotropically immobilized spin labels of the type seen in lipid and soap-like bilayers using a rigorous formalism which explicitly includes the effects of spin-label motion. In most bilayer systems, spin-label experiments have shown lower order parameters then deuterium-label experiments. In the past this apparent decrease in the order parameters was thought to reflect the distortion of the bilayer by the doxyl ring of the spin probes. We wish to report that this type of discrepancy may be due to the neglect of important motional effects in the time-independent effective Hamiltonian formalisms used in previous interpretations of anisotropically immobilized spin label spectra. That the true order parameters may be the same can be shown by including slow motional corrections in the effective Hamiltonian formalism. The larger volume of the doxyl ring may change the apparent order parameter by increasing the importance of the slow motional effects, as opposed to causing a real decrease in the order parameter, as previously proposed.

Molecular motion and order in single-bilayer vesicles and multilamellar dispersions of egg lecithin and lecithin-cholesterol mixtures. A deuterium nuclear magnetic resonance study of specifically labeled lipids.

Deuterium (2H) nuclear magnetic resonance (NMR) quadrupole splittings and relaxation times have been measured for a variety of specifically deuterated lipids intercalated in lamellar-multibilayer dispersions and single-bilayer vesicles of egg lecithin and lecithin-cholesterol mixtures. The deduced order parameters and relaxation times vary with position of deuteration, acyl chain length, unsaturation, and temperature. The order parameters and spinlattice relaxation times T1 indicate rapid intramolecular motions of restricted amplitude in both the choline head group and hydrocarbon chains. The ordering profile for the acyl chains is similar to that predicted by statistical-mechanical theory. The order parameters yield estimates of the bilayer thickness and linear coefficient of expansion in close agreement with the x-ray determinations. A comparison of the deuterium and electron spin resonance spinprobe order parameters demonstrates the perturbation of the bilayer by the bulky nitroxide probe. The transverse relaxation time T2 for single-bilayer vesicles is quantitatively accounted for by a simple modification of classical relaxation theory which takes into account the modulation of the static quadrupole interaction by rapid local molecular motions and the modulation of the residual quadrupole interaction by the slower overall tumbling of the vesicle. It is unambiguously demonstrated that molecular motion and order in single-bilayer vesicles are very similar to those in lamellar multibilayers. Significant differences occur only for a few segments near the terminal methyl groups of the acyl chains, where the order parameters for vesicles are 10-30% smaller than those found for lamellae. The incorporation of cholesterol in lecithin bilayers is shown to increase the degree of orientational order in vesicles and lamellae, and to increase the hydrodynamic radius of vesicles. Thus, single-bilayer vesicles and multilamellar dispersions of phospholipids are equally useful models for biological membranes. They yield equivalent information about the internal organization and mobility of lipid bilayers, when the spectral manifestations of overall vesicle motion are correctly taken into account.

Molecular order in Acholeplasma laidlawii membranes as determined by deuterium magnetic resonance of biosynthetically-incorporated specifically-labelled lipids.

The first application of deuterium magentic resonance of specifically labelled lipids to the study of a natural biological membrane is described. Palmitic acid labelled at the terminal methyl group with deuterium was incorporated biosynthetically into the lipids of the plasma membrane of Acholeplasma laidlawii. The deuterium nuclear magnetic resonance spectra contain quadrupole splittings which yield directly order parameters for this region of the membrane. Below the growth temperature (37 degrees C) the spectra are indicative of lipid in both gel and liquid crystalline states. Above this temperature they demonstrate the existence of an entirely liquid crystalline membrane whose order parameter decreases rapidly with increasing temperature. Comparison with egg phosphatidylcholine over the same temperature range shows a more rapid change in order with temperature for the A. laidlawii membranes.