Barney L. Bales

Inhomogeneously Broadened Spin-Label Spectra

Inhomogeneous broadening of the ESR lines of nitroxide spin labels due to hyperfine structure is a pest that has complicated the work of spin labelers since shortly after the beginning of the spin-label era (Plachy and Kivelson, 1967; Poggi and Johnson, 1970) continually until the present (for example, Jolicoeur and Friedman, 1971; Kovarskii et al, 1972; Bullock et al, 1975; Ahn, 1976; Freed, 1976; Lim et al, 1976; Jones and Schwartz, 1981; Ottaviani et al, 1983; More et al, 1984; Lee and Shetty, 1985). It is a problem that can and has been solved rigorously for a number of spin labels by combining NMR, ESR, and chemical substitution techniques (Kreilick, 1967; Briere et al, 1967, 1970; Michon and Rassat, 1971; Chiarelli and Rassat, 1973; Lim et al, 1976; Barbarin et al, 1978a, 1978b; Labsky et al, 1980; Windle, 1981; Ottaviani, 1987). These techniques taken together establish the hyperfine patternof a given spin label and ESR simulation fine tunes the hyperfine coupling constants in a given set of experimental conditions. To proceed with rigor, this fine tuning must be done continually because the coupling constants vary with temperature (Kreilick, 1967; Briere et al, 1967,1970; Jolicoeur and Friedman, 1971; Atherton, 1975; Ottaviani, 1987) and solvent (Briere et al, 1970; Freed, 1976; Lim et al, 1976; Eaton et al, 1980; Windle, 1981; Ottaviani, 1987). Also, the effectivespacing of the hyperfine lines varies with spin-label concentration—a variation that depends strongly on temperature (Plachy and Kivelson, 1967). The effective spacing varies with spin-label alignment in an ordered fluid (Polnaszek and Freed, 1975; Bales et al, 1984) and one can imagine that it might vary with other experimental parameters as well. Thus, the problem can be complicated but it can be solved with remarkable precision in some cases, notably with spin labels having particularly simple patterns or ones that give partially resolved ESR spectra. It is a tedious procedure, unavoidable if high precision is required in an experiment involving partially resolved ESR spectra. In most of the spin-label literature, the spectra are unresolved, either intrinsically by nature of the spin label and the experiment, or artificially, because of broadening due to oxygen or some other paramagnetic species. In unresolved spectra the procedures to correct for inhomogeneity become quite simple and remarkably accurate.

On cell membrane lipid fluidity and plant lectin agglutinability. A spin label study of mouse ascites tumor cells

The fluidity of the plasma membrane of Sarcoma 180 mouse ascites tumor cells has been studied in viable cells using fatty acid spin labels. The order parameter was found to vary from 0.61, approximately four carbon bond lengths removed from the membrane surface, to 0.47 approximately eleven bond lengths removed at 22 degrees C and from 0.55 to 0.33 at 37 degrees C. Thus these cells show similar membrane fluidity to that found in other mammalian cells with the exception of human erythrocytes which are less fluid. The concanavalin A mediated agglutinability of Sarcoma 180 cells was altered by the addition of cytochalasin B and the fluidity was found to be the same as in unaltered cells.

Correction for inhomogeneous line broadening in spin labels, II

Abstract Our methods to correct for inhomogeneous line broadening in the EPR of nitroxide spin labels are extended. Previously, knowledge of the hyperfine pattern of the nuclei responsible for the inhomogeneous broadening was necessary in order to carry out the corrections. This normally meant that either a separate NMR experiment or EPR spectral simulation was needed. Here a very simple method is developed, based upon measurement of four points on the experimental EPR spectrum itself, that allows one to carry out the correction procedure with precision rivaling that attained using NMR or spectral simulation. Two associated problems are solved: (1) the EPR signal strength is estimated without the need to carry out double integrations and (2) linewidth ratios, important in calculating rotational correlation times, are corrected. In all cases except one, the corrections are effected from the four measured points using only a hand-held programmable calculator. Experimental examples illustrate the methods and show them to be amazingly accurate.

Effect of the nature of the counterion on the interaction between cesium and tetraalkylammonium dodecylsulfates and poly(ethylene oxide) or poly(vinylpyrolidone).

The interaction between poly(ethylene oxide) or poly(vinylpyrrolidone) and cesium and tetraalkylammonium (tetramethyl to tetrabutyl ammonium) dodecylsulfate has been investigated by means of electrical conductivity measurements to determine the critical aggregation concentration (cac) of the surfactants in the presence of polymer. The cac values were compared to the values of the critical micellization concentration (cmc) of the surfactants in the absence of polymer. The value of the cac/cmc ratio increased with the radius of the counterion in the sequence: Na(+)<Cs(+)<tetramethylammonium(+)<tetraethylammonium(+)=tetrapropylammonium(+)=tetrabutylammonium(+)=1.0. This result indicates that the strength of the interaction decreases upon increasing counterion radius. For the last three tetraalkylammonium ions the value cac/cmc=1 indicates the absence of interaction. The results are discussed in an attempt to gain a better understanding of the mechanism of formation of surfactant aggregates bound to water-soluble polymers such as poly(ethylene oxide) or poly(vinylpyrrolidone).

DMPG gel–fluid thermal transition monitored by a phospholipid spin labeled at the acyl chain end

Low ionic strength aqueous dispersion of dimyristoyl phosphatidylglycerol (DMPG) presents a rather peculiar gel-fluid thermal transition behavior. The lipid main phase transition occurs over a large temperature interval (ca. 17 degrees C), along which several calorimetric peaks are observed. Using lipids spin labeled at the acyl chain end, a two-peak electron spin resonance (ESR) spectrum is observed along that temperature transition region (named intermediate phase), at three different microwave frequencies: L-, X- and Q-bands. The intermediate phase ESR spectra are analyzed, and shown to be most likely due to spin labels probing two distinct types of lipid organization in the DMPG bilayer. Based on the ESR spectra parameters, a model for the DMPG intermediate phase is proposed, where rather fluid and hydrated domains, possibly high curvature regions, coexist with patches that are more rigid and hydrophobic.

A simple, accurate method of correcting for unresolved hyperfine broadening in the EPR of nitroxide spin probes to determine the intrinsic linewidth and heisenberg spin exchange frequency

Abstract A method of correcting the EPR linewidth of a nitroxide that is inhomogeneously broadened by unresolved hyperfine structure is presented. Two simple equations are presented which allow the accurate deduction of the intrinsic linewidth and the Heisenberg spin exchange frequency, respectively, from the measured linewidths. The method allows correction for the inhomogeneous broadening in practically all cases in which the nitroxide tumbles in the motional narrowing region by using only a hand-held programmable calculator, thus eliminating the need for calibration libraries. The accuracy of the two equations is demonstrated for three nitroxides.

Matrix ENDOR Linewidths of Trapped Electrons in Glassy Matrices at 77°K

Matrix ENDOR lines of protons associated with trapped electrons in γ‐irradiated glassy matrices of 10M NaOH, methanol and 2‐methyltetrahydrofuran at 77°K have been observed. By analysis of the matrix ENDOR line shapes under comparable experimental conditions the linewidth has been related to the spatial extent of the ground state wavefunction of the trapped electron. These experimental results are compared with predictions of the semicontinuum model for trapped electrons and found to be in good agreement.

PROBING DMPG VESICLE SURFACE WITH A CATIONIC AQUEOUS SOLUBLE SPIN LABEL

A small, highly aqueous soluble, deuterated, cationic spin label, 4-trimethylammonium-2,2,6,6-tetramethylpiperidine-d17-1-oxyl iodide (dCAT1), was used to directly monitor the negatively charged DMPG vesicle surface in order to test a recent suggestion (Riske et al., Chem. Phys. Lipids, 89 (1997) 31-44) that alterations in the surface potential accompanied apparent phase transitions observed by light scattering. The temperature dependence of the label partition between the lipid surface and the aqueous medium indicated an increase in the surface potential at the gel to liquid-crystal transition, supporting the previous suggestion. Results at the phase transition occurring at a higher temperature were less definitive. Although some change in the dCAT1 ESR spectra was observed, the interpretation of the phenomena is still rather unclear. DMPG surface potentials were estimated from the dCAT1 partition ratios (surface label moles/total label moles), using a simple two-sites model, where the electrostatic potential is zero everywhere but at the vesicle surface, and the interaction between the spin label and the membrane surface is chiefly electrostatic. The Gouy-Chapman-Stern model predicts surface potentials similar to those observed, although the measured decrease in the surface potential with ionic strength is somewhat steeper than that predicted by the model.

EPR investigation of the intermediate spin exchange regime

The electron paramagnetic resonance spectra of di‐tert butylnitroxide (DTBN) are studied as a function of the nitroxide concentration in three hydrocarbon solvents at constant temperature. In the concentration range studied, DTBN undergoes electron spin exchange which leads to broadening, shifting, and eventual collapse of the lines forming the 19‐line proton hyperfine pattern. Particular emphasis is given to the spin exchange frequencies ωHE in the region in which they are comparable with the proton hyperfine coupling constants—the intermediate spin exchange regime. Both linewidths and line shapes are found to be in full accord with theory in the intermediate exchange regime which completes the demonstration that theory and experiment are in agreement, the slow and fast exchange regimes having been studied many times in the past. Modeling the spectral lines as Lorentzian–Gaussian convolutions allows a detailed exposition of the collapse of the hyperfine structure as ωHE increases and this collapse, too, ...

EPR Line Shifts and Line Shape Changes due to Spin Exchange of Nitroxide Free Radicals in Liquids: 6. Separating Line Broadening due to Spin Exchange and Dipolar Interactions

EPR spectra of perdeuterated 2,2,6,6-tetramethyl-4-oxopiperidine-1-oxyl (PDT) are studied as functions of molar concentration, c, and temperature, T, in water and 70 wt % glycerol in water. The increase of the intrinsic line width averaged over the three hyperfine lines, B(tot), varies linearly with c with zero intercept in both solvents at all temperatures; therefore dB(tot)/dc is independent of c. The spin exchange induced dispersion, from which the spin exchange frequency, omega(e), may be computed, increases linearly with B(tot), passing through the origin in water and in 70% glycerol at high temperatures; however, at low temperatures, where dipolar interactions broaden the spectra, linearity does not prevail until B(tot) > 1 G due to a contribution of dipolar interactions to the dispersion. The broadening constant due to spin exchange, dB(e)/dc, is found from the slope of the linear region, permitting a computation of the dipolar constant, dB(dip)/dc = dB(tot)/dc - dB(e)/dc. Thus, the separation of concentration broadening into spin exchange and dipolar contributions is effected without having to appeal to some supposed temperature dependence of the two interactions. The fractional broadening by spin exchange, Omega(T), is near unity at high temperatures in both solvents, decreasing to zero in 70% glycerol at 273 K. Omega(T) is a continuous function of the inverse rotational correlation time of PDT but is discontinuous as a function of T/eta where eta is the shear viscosity. Omega(T) = 0.5, where spin exchange and dipolar interactions contribute equally to the line width occurs at T/eta = 20 +/- 1 K/cP in 70% glycerol. Hydrodynamic predictions of dB(e)/dc via the Stokes-Einstein (SE) equation are remarkably accurate in 70% glycerol comparable with the results in a series of alkanes. In water, dB(e)/dc is linear with T/eta with zero intercept as required by the SE; however, with slope a factor of 0.73 smaller. dB(dip)/dc is reasonably predicted by the SE only at very small values of eta/T very quickly following an approximately logarithmic dependence rather that the linear prediction. Values of dB(dip)/dc approach a plateau above eta/T = 0.20 cP/K that is about one-half the solid state limit. Line shifts due to spin exchange are not yet useful to deduce values of Omega(T) due to a lack of knowledge of the time between re-encounters; however, they may be used to verify the values determined from line broadening and spin exchange induced dispersion. Some effects at low temperatures in 70% glycerol suggest that the effects of dipolar interaction are inadequately described by the widely accepted theory.