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[Distribution of the tempo-dichlorotriazine spin label in the immunoglobulin molecule].

Spin-label 14C-TEMPO-dichlorotriazine distribution in Fab- and Fc-fragments of monoclonal myeloma immunoglobulin IgG (lambda) 1 Bel, in their heavy and light chains, and tryptic fragments of chains was investigated. Radio-active spin label in proteolytic Fab- and Fc-fragments, in heavy and light chains, and in separate peptides was detected. 3 cm and 2 mm band ESR spectra of spin-labeled IgG in water solutions was studied. It was shown that the models of highly anisotropic motion, isotropic motion and slow isotropic motion of spin label around globule or the description of experimental ESR spectra of TEMPO-dichlorotriazine spin-labeled immunoglobulins do not work.

Cyclic N-Oxides↗
PubMed1994

Oxygen production and consumption by chloroplasts in situ and in vitro as studied with microscopic spin label probes.

A new spin-label oximetry approach able to measure the oxygen partial pressure in complex photosynthetic systems has been developed using bovine serum albumin (BSA)-coated light paraffin oil particles containing cholestane spin label (CSL). Paraffin oil particles protect the spin label against the action of chemically active metabolites. The amplitude of the electron paramagnetic resonance (EPR) signal from CSL measured at a saturating microwave power is sensitive to the concentration of oxygen. We demonstrate here the ability of this method to monitor the kinetics of light-induced oxygen production in situ, i.e., in the interior of a bean leaf. The oxygen release, observed during leaf illumination with continuous light, exhibits an overshoot that correlates with the well-known nonmonotonous behaviour of the Photosystem I reaction center, P700. Short-term illumination of isolated bean chloroplasts, suspended in the presence of the electron mediator methylviologen, induces a reversible uptake of oxygen. However, after prolonged illumination, chloroplasts lose their ability to regenerate oxygen in the dark. The exhaustion of oxygen (and oxygen active forms) is accompanied by the loss of CSL paramagnetism and the capacity to photooxidize P700. Comparison of the kinetics of P700 redox transients with oximetric data demonstrates that oxygen concentration is the essential factor controlling electron transport in leaves and isolated chloroplasts.

Chlorophyll↗
PubMed1994

Studies of the substrate binding to xanthine oxidase using a spin-labeled analog.

A spin-labeled adenine derivative [N6-(2,2,6,6-tetramethyl-1-oxypiperidin-4-yl)adenine; SLAD] is found to be a very slow substrate of xanthine oxidase based on the observed reduction of enzyme by SLAD under anaerobic conditions. A room-temperature EPR spectrum of SLAD in the presence of oxidized xanthine oxidase shows the appearance of "wings" on the three-line spectrum of the free spin-label, indicating formation of an E.SLAD complex. This spectrum can be obtained on a timescale that is short compared to catalysis. Using this spectral change as an experimental probe, the room-temperature Kd's of SLAD binding to oxidized xanthine oxidase at various pH's have been determined. Obtained Kd values are 1.5 +/- 0.3 mM, 1.6 +/- 0.3 mM, and 1.5 +/- 0.3 mM at pH 10.0, 8.5, and 7.0, respectively, indicating no significant difference in the equilibrium dissociation of SLAD from enzyme upon pH change. These results are consistent with the calculated equilibrium dissociation constant for substrate binding to oxidized molybdenum center based on Kd to reduced enzyme and the perturbation of MoVI/MoV and MoV/MoIV reduction potentials by product and substrate analogs.

Adenine↗
PubMed1987

Radiolysis of spin-labeled DNA: an electron spin resonance investigation.

The reactions of free and DNA-bound 2,2,5,5-tetramethylpyrrolidine-N-oxyl (PROXYL) probes with radicals generated during radiolysis of dilute aqueous solutions of DNA were examined. For the free PROXYL probe in deaerated solution with each of the four nucleotides (dAMP, dCMP, dGMP, and TMP) it was found that the pyrimidine radicals were more reactive toward the probe than were the purine radicals. Reactions of the electron adduct of TMP and the hydroxyl radical adducts of dAMP, dGMP, and TMP with the probe resulted in little or no reduction of the probe. For TMP these results are consistent with the fact that both the protonated electron and hydroxyl radical adducts of TMP will covalently bind to the nitroxide function of the probe. Reduction of the PROXYL probe was observed in reactions with the hydroxyl radical adduct of dCMP and with the electron adducts of dAMP, dCMP, and dGMP. Results of the radiolysis of the free PROXYL probe in deaerated dilute solution of DNA suggest that the PROXYL probe protects the DNA from water radical attack as the ratio of DNA bases to PROXYL probe increases above 50:1. Reactions of DNA-bound probes are dependent on the depth of the nitroxide function in relation to the major groove of the DNA helix. Two probes with tether lengths which are less than the depth of the major groove show an expected increase in reactions with DNA base radicals as compared to a probe with a tether that extends beyond the groove. The longer probe is involved largely in reactions with sugar and water radicals along the periphery of the DNA helix. In the presence of oxygen, there is a dramatic decrease in the loss of both the free and DNA-bound probes due to the lack of reaction of these probes with peroxyl radicals formed by the addition of molecular oxygen to DNA radicals.

Cyclic N-Oxides↗
PubMed1982

The role of the nicotinamide moiety of NAd+ for negative cooperativity in glyceraldehyde-3-phosphate dehydrogenase as studied by spin-labeled cofactors.

Two derivatives of NAD+ spin-labeled at N6 or C-8 of the adenine ring have been shown previously to be active coenzymes of glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12). When more than two equivalents of either spin-labeled NAD+ are bound to the tetrameric enzyme, spin-spin interaction is observed in the ESR spectra (Deparade, M.P., Glöggler, K. and Trommer, W.E. (1981) Biochim. Biophys, Acta 659, 422-433). After reduction of enzyme-bound NAD+ spin-labeled at C-8 to the corresponding NADH derivative, the additional peaks due to this spin-spin interaction disappear, which implies that the distance between the two radicals increases. It is proposed that the coenzyme slide further towards the active site upon reduction. ADPR spin-labeled at C-8 binds non-cooperatively, exhibiting a dissociation constant of Kd = 33 microM. Even with 3.5 equivalents bound to the enzyme, spin-spin interaction is not observed. AMP spin-labeled at C-8 combines with two sites per monomer, or a total of eight per tetramer. The respective dissociation constants are Kd1 = 30 microM and Kd2 - 2.3 mM. Phosphate competes with AMP bound to the weak site. Spin-spin interaction is not observed. ATP spin-labeled at C-8 is bound about 10-fold tighter than the corresponding AMP derivative. Four equivalents of ATP are bound per tetramer, but it exhibits no spin-spin interactions. It is concluded that the structure of the pyridine moiety of the coenzymes plays a role in orienting the adenine ring and, thus, affects the cooperativity. The N6 derivative of NAD+ also shows spin-spin interaction; however, only data for the C-8 derivatives are shown in detail.

Animals↗
PubMed1990

Comparison of Ca(II), Cd(II), and Mg(II) titrations of tyrosine-99 spin-labeled bovine calmodulin.

Bovine calmodulin, spin-labeled at tyrosine-99, has been utilized in electron paramagnetic resonance (EPR) studies to investigate calmodulin interactions with Ca(II), Cd(II), and Mg(II). The addition of either Ca(II) or Cd(II) to apo-calmodulin results in a complex capable of activating target enzymes, such as 3', 5'-cyclic nucleotide phosphodiesterase (J. M. Buccigross, C. L. O'Donnell, and D. J. Nelson, Biochem. J. 235 677 [1986]), while Mg(II) is known to be incapable of activating calmodulin toward any of its target enzymes. Additions of Ca(II) and Cd(II) to spin-labeled apo-calmodulin gave rise to very similar changes in the EPR spectrum of the bound label, consistent with a dramatic decrease in the mobility of the nitroxide spin-label covalently attached to tyrosine-99. Addition of Mg(II) to spin-labeled apo-calmodulin caused no change in the EPR spectrum of the bound label. Thus, the conformational changes induced by Ca(II) and Cd(II) ion binding to calmodulin, which lead to decreased tyrosine-99 spin label mobility, are clearly not occurring when Mg(II) ion binds. These results are consistent with the results of other spectroscopic studies, which indicate that "activating" metal ions, such as Ca(II) and Cd(II), produce calmodulin conformers that are different from those produced by "inactivating" metal ions, such as Mg(II).

Animals↗
PubMed1976

Lanthanum and some other cation-induced changes in fluidity of synaptosomal membrane studied with nitroxide stearate spin labels.

Using nitroxide fatty acid spin labels, the effects of some cations such as La3+, Cd2+ and Hg2+ on synaptosomal membranes were studied by observing changes in their ESR spectra. The labels were incorporated almost instantaneously into synaptosomes isolated from rat brain cortex. ESR spectra of the spin-labeled synaptosomes were significantly braodened immediately upon adding La3+, Ce3+, Cd2+ or Hg2+ but hardly affected by Ca2+, Sr2+ and Ba2+. The magnitude of the change in the separation of the outer two peaks in ESR spectra (2T') depends on the number (n) of methylene units between the polar head group and the spin-label (nitroxide) group; that is, it increases with decreasing n. Among these ions, the effect of La3+ was the greatest and appeared to be in parallel with the amount of La3+ bound with the synaptosomes. On the other hand, K+, Rb+ or Li+ causes hardly any significant changes.

Animals↗
PubMed1997

Global topology & stability and local structure & dynamics in a synthetic spin-labeled four-helix bundle protein.

A maleimide nitroxide spin-label (MAL-6) linked to a cysteine in the hydrophobic core and a coproporphyrin I (CP) appended on the N-terminus of a synthetic helix-loop-helix peptide ([alpha2]) have been used to examine the designed self-association of a four-helix bundle ([alpha2]2), focusing on the bundle topology and stability and the rotational dynamics of the spin-label. Gel-permeation chromatography demonstrated that the [alpha2] peptide and the peptide modified with a spin-label ([MAL-6-alpha2]), a coproporphyrin ([CP-alpha2]) and a coproporphyrin plus a spin-label ([CP-MAL-6-alpha2]) self-associate into four helix bundles in solution as designed. Circular dichroism (CD) spectra prove that all these peptides are highly alpha-helical, confirmed for [alpha2]2 by Fourier transform infrared (FTIR) spectroscopic analysis. Electron spin resonance (ESR) spectra of the two attached maleimide spin-labels in [MAL-6-alpha2]2 shows their effective rotational correlation time (tau(c)) is 7.3 +/- 0.5 ns, consistent with that expected for the tumbling of the four helix bundle itself, indicating the labels are immobilized. The ESR spectra were also unaltered by aqueous-phase paramagnetic ions, Ni(II), demonstrating all of the spin-labels are buried within the hydrophobic core. The lack of spin-spin interaction between the buried, immobilized spin-labels indicates they are remote (> 15 A) from each other, indicating an antiparallel topology of the monomers in [MAL-6-alpha2]2. The parent [alpha2]2 and the modified [MAL-6-alpha2]2 and [CP-alpha2]2 peptides are highly stable (deltaG(H2O) approximately 25 kcal/mol) as investigated by guanidine hydrochloride denaturation curves monitored by ESR and CD spectroscopies. Guanidine hydrochloride denaturation leads to a shorter correlation time of the spin-label, tau(c) < 1 ns, approaching that of an unrestricted spin-label in solution. In contrast, trifluoroethanol caused dissociation of [MAL-6-alpha2]2 to yield two [MAL-6-alpha2] monomers with retention of secondary structure and changed the tau(c) to 2.5 +/- 0.5 ns, indicating that a significant degree of motional restriction is imposed on the spin-label by the secondary structure. The coproporphyrin probes covalently attached to the N-termini of [CP-alpha2]2 and [CP-MAL-6-alpha2]2 provided evidence that the helical monomers of both were in a parallel orientation, in contrast to the antiparallel orientation determined for [MAL-6-alpha2]2. Consequently, the ESR spectra of [MAL-6-alpha2]2 and [CP-MAL-6-alpha2]2 reveal major structural differences in the local vicinity of the spin-labels due to the topological difference between these two bundles. The ESR spectra of [CP-MAL-6-alpha2]2 contains two distinct nitroxide populations, indicating that one spin-label remains buried in the hydrophobic core and the other is excluded to solvent in this parallel topology. Alleviation of the steric interactions causing one spin-label in [CP-MAL-6-alpha2]2 to be solvent-exposed by addition of [CP-alpha2]2 results in formation of the heterodimeric [CP-alpha2]/[CP-MAL-6-alpha2], as evidenced by insertion of all the spin-labels into hydrophobic cores. The changes in global topology and local structure as evidenced by this pair of spectral probes have relatively minor effects on the course of guanidine denaturation of these bundles.

Amino Acid Sequence↗
PubMed1986

Microenvironment around the essential cysteine residues in chicken liver fructose-1,6-bisphosphatase as analyzed by ESR spin labelling.

Chemical modification and electron spin resonance spectroscopy (ESR) spin-labelling techniques have been employed to investigate the local environment of the essential sulfhydryl groups of chicken liver fructose-1,6-bisphosphatase. The results demonstrate the presence of two distinct classes of sulfhydryl groups in this enzyme. The first class react preferentially with iodoacetate and its spin-labelled derivative, and this results in an increase in catalytic activity, while the second class react preferentially with N-ethylmaleimide and its spin-labelled derivative, and this leads to a decrease in catalytic activity. The ESR spectral data strongly suggest that the first class of sulfhydryl groups are located in a deep cleft of the enzyme molecule, while the second class of sulfhydryl groups are located in a shallow crevice. The environment of the second class of the sulfhydryl groups appears to undergo a significant change after the modification of the first class of sulfhydryl groups by iodoacetate.

Animals↗
PubMed1981

Distinct states of lipid mobility in bovine rod outer segment membranes. Resolution of spin label results.

Freely diffusable lipid spin labels in bovine rod outer segment disc membranes display an apparent two-component ESR spectrum. One component is markedly more immobilized than that found in fluid lipid bilayers, and is attributed to lipid interacting directly with rhodopsin. For the 14-doxyl stearic acid spin label this more immobilized component has an outer splitting of 59 G at 0 degrees C, with a considerable temperature dependence, the effective outer splitting decreasing to 54 G at 24 degrees C. Spin label lipid chains covalently attached to rhodopsin can also display a two-component spectrum in rod outer segment membranes. In unbleached, non-delipidated membranes the 16-doxyl stearoyl maleimide label shows an immobilized component which has an outer splitting of 59 G at 0 degrees C and a considerable temperature dependence. This component which is not resolved at high temperatures (24--35 degrees C), is attributed to the lipid chains interacting directly with the monomeric protein, as with the diffusable labels. In contrast, in rod outer segment membranes which have been either delipidated or extensively bleached, a strongly immobilized component is observed with the 16-doxyl maleimide label at all temperatures. This immobilized component has an outer splitting of 62--64 G at 0 degrees C, with very little temperature dependence (61--62 G at 35 degrees C), and is attributed to protein aggregation.

Animals↗
PubMed1976

Specific spin-labeling of transfer ribonucleic acid molecules.

The spin labels anhydride (ASL), bromoacetamide (BSL) and carbodiimide (CSL) were used to label selectively tRNAGlu, tRNA fMet and tRNAPhe from E. coli. The preparation and characterization of the sites of labeling of eight new spin-labeled tRNAs are described. The sites of labeling are: s2U using ASL, BSL and CLS and tRNAGlu; s4U using ASL and BSL on tRNAfMet and tRNAPhe; U-37 with CSL on tRNfMet; U-33 with CSL on tRNAPhe. The rare base X at position 47 of tRNAPhe has been acylated with a spin-labeled N-hydroxysuccinimide (HSL). The 3'end of unfractionated tRNA molecules has been chemically modified to a morpholino spin-labeled analogue (MSL). Their respective e.s.r. spectra are reported and discussed.

Binding Sites↗
PubMed1996

Preparation and characterization of a biologically active spin-labeled sea anemone toxin.

A derivative of the polypeptide cardiostimulant anthopleurin-B(AP-B) labeled with the spin label 1-oxyl 2,2,6,6-tetramethyl-4-piperidinyloxycarbonyl azide has been prepared and characterized. The product was found by mass spectrometry to be labeled at a single site, which amino acid sequencing showed to be the N-terminus. It also retained positive inotropic activity when assayed on isolated guinea pig atria. The spin-labeled (SL) product was found to exist in two distinct conformations by reversed-phase HPLC and in at least two conformations by electron spin resonance spectroscopy (ESR) over the pH range 2-9. The ESR data also show evidence for multimetric states of SL-AP-B over the pH range 2-9, with maximum aggregation at pH 4.5-5, and a slow disaggregation when the pH is adjusted to 8-9. The presence of multiple conformers of SL-AP-B and its tendency to aggregate render it unsuitable for high-resolution NMR structural studies of the isolated ligand, but the retention of activity may make it useful for studies of the sodium-channel-bound form of the molecule.

Animals↗
PubMed1990

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)

Anesthetics↗
PubMed1988

Binding of spin-labeled clofibrate to lipoproteins.

The binding of spin-labeled clofibrate to native and partially delipidated lipoproteins is a rapid, linear and non-saturable process observed up to the critical micellar concentration of the drug. Low-density lipoproteins (LDL) display a lower affinity for the drug than very-low-density lipoproteins (VLDL) and high-density lipoproteins (HDL) relative to their respective specific volume. Unlike various lipophilic drugs, uptake of spin-labeled clofibrate does not correlate with lipoprotein lipid volume. Spin-labeled clofibrate binding to LDL is enhanced when the temperature increases above 25 degrees C. The binding to HDL and VLDL is less temperature-sensitive. The simulation of the ESR spectra has shown that two types of motion should be superimposed for the spin-labeled clofibrate in HDL, in LDL or in partially delipidated LDL. From 40 down to 25 degrees C for HDL and LDL, a fast anisotropic motion is observed. From 25 degrees C down to 5 degrees C, a two-component motion takes place, including a slow isotropic motion of the probe tumbling in a highly hydrophobic environment. Interactions of spin-labeled clofibrate with the apolipoproteins in HDL and LDL are assumed from the emergence of this strongly immobilized component observed when the temperature decreases. In contrast, for spin-labeled clofibrate inserted in the apolar core of VLDL, ESR shows only one component in the whole temperature range (5-40 degrees C). The location of the spin-labeled drug inside the various lipoprotein particles is discussed as a function of temperature.

Clofibrate↗
PubMed1993

Aggregation state of spin-labeled cecropin AD in solution.

A spin-labeled derivative of the ion channel peptide cecropin AD (Fink et al., 1988) was synthesized and used to investigate its aggregation state in water and in the presence of a helix-promoting solvent. A cysteine was introduced at position 33 and spin-labeled using the methanethiosulfonate spin label. In low ionic strength aqueous solution, the peptide is monomeric, and the ESR spectrum indicates a high degree of segmental flexibility at the nitroxide attachment point, consistent with a predominantly random coil conformation. Upon addition of 5-10% (v/v) hexafluoro-2-propanol (HFP), the peptide is induced to aggregate as evidenced by significant motional restriction of the spin label and spin-spin broadening of the ESR lines. At higher concentrations of HFP, the peptide reverts to a monomeric state but retains its folded conformation. Our data suggest that between 5 and 10% HFP the peptide undergoes two structural transitions. The first transition starts at 5% and is very cooperative. Its dependence on ionic strength, temperature, and pH indicates that it involves the interconversion between a random coil and an ordered state stabilized by interpeptide electrostatic and hydrophobic interactions. The second transition, which occurs at 11% v/v HFP, is between the self-associated form and an ordered monomeric form. The analysis of our experimental results demonstrates aggregate formation at 5-10% HFP. This may be relevant to the mechanism of channel formation by cecropins in membranes.

Amino Acid Sequence↗
PubMed1980

Analysis of phosphoryl transfer mechanism and catalytic centre geometries of transport ATPase by means of spin-labelled ATP.

Spin-labelled ATP [3'-O-(1-oxyl-2,2,5,5-tetramethyl-3-carbonyl pyrrolidine)-adenosine 5'-triphosphate], abbreviated SL-ATP, is used to study firstly the occurrence of an associative phosphorane mechanism for the phosphoryl transfer from ATP to the transport-ATPase protein, and secondly the presence of two geometrically unequal catalytic centres in the two catalytic peptide chains deduced to explain the existence of two KD'(ATP) values under equilibrium conditions and two Km(ATP) values under turnover conditions. 1. In the presence of Na+, K+ and Mg2+, SL-ATP is not hydrolysed by transport-ATPase from three different sources. In the presence of Na+ and Mg2+, SL-ATP reacts initially like ATP with the enzyme, as indicated by the production of a similar ouabain-binding protein conformation. With both nucleotides, this initial reaction includes the formation of the covalent enzyme-nucleotide complex through nucleophilic attack of the aspartate carboxyanion of the catalytic centre on the terminal phosphorus atom of the triphosphate chain. This produces the ouabain-binding conformation of the enzyme. Unlike ATP, the covalent enzyme-SL-ATP complex resists further transformation. 2. In the presence of Na+, K+ and Mg2+, the influence of SL-ATP on ATP hydrolysis by transport-ATPase depends on the ATP concentration chosen. At low ATP concentration, when the enzyme works as Na+-ATPase, SL-ATP does not affect the rate of ATP cleavage. At high ATP concentration, however, when the enzyme works as (Na+ + K+)-ATPase, SL-ATP reduces the rate of ATP hydrolysis to the level of Na+-ATPase activity, apparently due to the formation of the covalent enzyme-SL-ATP complex. 3. SL-ATP in the covalent enzyme-SL-ATP complex shows an ESR spectrum which is indistinguishable regarding the overall shape, the rotational correlation time, tau, and the hyperfine coupling constant, aN, from the ESR spectrum of free SL-ATP. Consequently, the dimensions of the catalytic centre cleft of transport-ATPase provide the labelled group of SL-ATP, opposite to its 3'-O-esterification site at the ribose moiety, in a wide-cleft groove, enough free space for an essentially unhindered rotational mobility within an aqueous environment like that of the bulk medium. Judged from literature data, similarly wide grooves exist in the catalytic centre clefts of mitochondrial and myosin ATPases. 4. In the framework of present knowledge, the idea is put forward that the structural unit forming the binding site for the AMP moiety of ATP in ATPases is similar to the structural unit forming the binding site for the AMP moiety of NAD and ADP in several dehydrogenases and kinases.

Adenosine Triphosphatases↗
PubMed1981

Apparent hydrogen bonding by strongly immobilized spin-labels.

The hyperfine separations of nitroxide spin-labels which are tightly bound within hemoglobin exhibit a substantial temperature dependence even when the hemoglobin is immobilized by freezing or precipitation. It is shown that NO.--HX hydrogen bond formation by the spin-label within its binding site is a good explanation for the observed temperature dependence. Comparative studies using different hemoglobin derivatives and two different spin-labels suggest that the HX group may be some element of the protein matrix and that this hydrogen bond may be a factor in the stabilization of the label within its binding site. The hyperfine separation of a fatty acid spin probe incorporated into aqueous bilayer dispersons of dipalmitoylphosphatidylcholine also exhibits a temperature dependence at low temperature which is qualitatively similar to that of the spin-labeled hemoglobin systems. Saturation transfer electron paramagnetic resonance measurements indicate that label motion is not the source of this temperature dependence. A hydrogen-bond equilibrium between water molecules and the nitroxide NO. group appears to be a plausible source of the temperature-dependent hyperfine separation in the lipid bilayer system. Small amplitude torsional oscillation or librational motion by the nitroxide may also produce additional changes in the hyperfine separation which are difficult to distinguish from hydrogen-bonding effects under some circumstances. The apparent hydrogen-bond equilibrium exhibits a strong thermal and environmental dependence which may be of importance in a number of biophysical spin-label measurements.

Electron Spin Resonance Spectroscopy↗