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PubMed1995

Selective labeling of membrane protein sulfhydryl groups with methanethiosulfonate spin label.

Electron paramagnetic resonance was used to characterize the first use of a thio-specific spin label in membranes. Procedures for use of the spin-label, 1-oxyl-2,2,5,5-tetramethyl-delta 3-pyrroline-3-methyl (methanethiosulfonate MTS) covalently attached to membrane proteins in human erythrocyte membranes are reported. The major findings are: (1) MTS was found to be thiol-specific in membranes as it is for soluble proteins; (2) MTS labels ghost proteins in as few as 30 min at room temperature, providing a distinct advantage when sensitive or fragile membranes are to be used; (3) the distribution of the spin label suggests that the major cytoskeletal protein, spectrin, and the major transmembrane protein (Band 3) incorporate the highest percentage of spin label. This procedure expands the tools with which the researcher can investigate the physical state of membrane proteins and its alteration upon interaction of membrane perturbants or in pathological conditions.

Acetylcholinesterase↗
PubMed2006

Motional restrictions of membrane proteins: a site-directed spin labeling study.

Site-directed mutagenesis was used to produce 27 single cysteine mutants of bacteriophage M13 major coat protein spanning the whole primary sequence of the protein. Single-cysteine mutants were labeled with nitroxide spin labels and incorporated into phospholipid bilayers with increasing acyl chain length. The SDSL is combined with ESR and CD spectroscopy. CD spectroscopy provided information about the overall protein conformation in different mismatching lipids. The spin label ESR spectra were analyzed in terms of a new spectral simulation approach based on hybrid evolutionary optimization and solution condensation. This method gives the residue-level free rotational space (i.e., the effective space within which the spin label can wobble) and the diffusion constant of the spin label attached to the protein. The results suggest that the coat protein has a large structural flexibility, which facilitates a stable protein-to-membrane association in lipid bilayers with various degrees of hydrophobic mismatch.

Amino Acid Substitution↗
PubMed1985

Lipid-protein interactions in frog rod outer segment disc membranes. Characterization by spin labels.

Freely-diffusing phospholipid spin labels have been employed to study rhodopsin-lipid interactions in frog rod outer segment disc membranes. Examination of the ESR spectra leads us to the conclusion that there are two motionally distinguishable populations of lipid existing in frog rod outer segment membranes over a wide physiological temperature range. Each of the spin probes used shows a two-component electron spin resonance (ESR) spectrum, one component of which is motionally restricted on the ESR timescale, and represents between 33 and 40% of the total integrated spectral intensity. The second spectral component which accounts for the remainder of the spectral intensity possesses a lineshape characteristic of anisotropic motion in a lipid bilayer, very similar in shape to that observed from the same spin labels in dispersions of whole extracted frog rod outer segment lipid. The motionally restricted spectral component is attributed to those spin labels in contact with the surface of rhodospin, while the major component is believed to originate from spin labels in the fluid lipid bilayer region of the membranes. Calculations indicate that the motionally restricted lipid is sufficient to cover the protein surface. This population of lipids is shown here and elsewhere (Watts, A., Volotovski, I.D. and Marsh, D. (1979) Biochemistry 18, 5006-5013) to be by no means rigidly immobilized, having motion in the 20 ns time regime as opposed to motions in the one nanosecond time regime found in the fluid bilayer. Little selectivity for the motionally restricted population is observed between the different spin-labelled phospholipid classes nor with a spin-labelled fatty acid or sterol.

Animals↗
PubMed2005

Application of molecular dynamics simulations to spin-labeled oligonucleotides.

The EPR study of spin labeled macromolecules has provided insight into structural and dynamical properties of DNA, proteins, and related systems. While spin labeling has been useful, it is experimentally difficult to determine if the spin label significantly alters the conformation of the macromolecule to which it is attached. Molecular modeling has proven to be a powerful tool for studying structure and dynamics of biologically important molecules. Here, we have conducted molecular dynamics (MD) studies of spin labeled oligonucleotides (ONs) bearing a five (5sp) or six (6sp) membered ring nitroxide, and the corresponding unmodified ON using the suite of programs contained in Amber 5.0 with the Cornell et al. 94 force field (Cornell, W. D., Cieplak, P., Bayly, C. I., Gould, I. R., Merz, Jr., K. M. Ferguson, D. M., Spellmeyer, D. C., Fox, T., Caldwell, J. W., and Kollman, P. A. A Second Generation Force Field for the Simulation of Proteins and Nucleic Acids. J. Am. Chem. Soc. 117, 5179-5197 (1995)). Quantum mechanical calculations employing the B3LYP method with the standard 6-31G* basis set using Gaussian98 were performed and, together with available crystallographic data for analogous nitroxides, new parameters for the nitrogen, oxygen, nitroxide alpha-carbon, and sp-hybridized carbon atoms have been developed suitable for the Cornell et al. 94 force field. MD simulations on the double-stranded (ds) spin labeled ONs, along with the corresponding unmodified analogues, have been studied over the course of 4 ns and conformational properties of all ONs are described based on the analysis of the trajectories. The spin labels were found to alter the global conformation of the ONs to which they were attached to accommodate the spin labels. The major changes include widening the major groove, decreasing helical twist, and more negative X-displacement of the base pairs. The magnitude of the effect was dependent on the specific structure of the spin label. Average and 'most representative' structures derived from the molecular dynamics simulations correlate with the experimental data on the spin labeled ONs.

Computer Simulation↗
PubMed1991

Dynamics and aggregation of the peptide ion channel alamethicin. Measurements using spin-labeled peptides.

Two spin-labeled derivatives of the ion conductive peptide alamethicin were synthesized and used to examine its binding and state of aggregation. One derivative was spin labeled at the C-terminus and the other, a leucine analogue, was spin labeled at the N-terminus. In methanol, both the C and N terminal labeled peptides were monomeric. In aqueous solution, the C-terminal derivative was monomeric at low concentrations, but aggregated at higher concentrations with a critical concentration of 23 microM. In the membrane, the C-terminal label was localized to the membrane-aqueous interface using 13C-NMR, and could assume more than one orientation. The membrane binding of the C-terminal derivative was examined using EPR, and it exhibited a cooperativity seen previously for native alamethicin. However, this cooperativity was not the result of an aggregation of the peptide in the membrane. When the spectra of either the C or N-terminal labeled peptide were examined over a wide range of membrane lipid to peptide ratios, no evidence for aggregation could be found and the peptides remained monomeric under all conditions examined. Because electrical measurements on this peptide provide strong evidence for an ion-conductive aggregate, the ion-conductive form of alamethicin likely represents a minor fraction of the total membrane bound peptide.

Alamethicin↗
PubMed1977

Synthesis and micellar formation of spin-labeled lysolecithin.

A spin-labeled lysolecithin, 1-[12'-(N-oxyl-4",4"-dimethyloxazolidine)-stearoyl]-sn-glycero-3-phosphorylcholine, has been synthesized in which the spin is covalently attached to its fatty acyl chain. The electron spin resonance spectra of this lysolecithin is an aqueous solution generally showed sharp three resonance lines superposed on a broad resonance line. Investigation of changes in the signal intensity of these spectra against the concentration of lysolecithin led to the inference that the sharp lines are due to monomers of lysolecithin while the broad one to micelles. The critical micellar concentration was consistent with that evaluated from the spectral shift of a dye. In the electron spin resonance spectra obtained from spin-labeled lysolecithin solutions with various amounts of dimyristoyllecithin, the line width of broad signal arised from micellar spin-labeled lysolecithin broadened as increase of dimyristoyllecithin. The line-broadening thus observed was briefly discussed.

Colloids↗
PubMed1986

Changes in conformation of spin-labeled calmodulin by phospholipids.

Spin-labeled calmodulin was synthesized and the effects of phospholipids on its conformation were examined by ESR spectroscopy. Phosphatidylserine (0.1-1.0 mM) increased the signal intensity of the ESR spectrum of spin-labeled calmodulin and decreased the apparent rotational correlation time in the presence of 0.1 mM CaCl2. This change was reversed by addition of excess calcium, and in the absence of calcium phosphatidylserine did not change the spectrum, suggesting that the change in spin-labeled calmodulin brought about by phosphatidylserine was not induced by a hydrophobic interaction of the two, but by inhibition of the binding of calcium to calmodulin. L-Serine and O-phospho-L-serine had no effect on the ESR signals of spin-labeled calmodulin. The effects of various other phospholipids were also examined. Their inhibitory activities were in the order phosphatidic acid greater than phosphatidylserine greater than phosphatidylglycerol = phosphatidylinositol; phosphatidylethanolamine and phosphatidylcholine had no effect on the spectra. The effects of these phospholipids were dependent on their binding activities toward calcium. Furthermore, phosphatidic acid and phosphatidylserine at 1 mM reduced the activity of calmodulin-dependent phosphodiesterase by 16.4 and 8.7%, respectively. These findings indicate that spin-labeled calmodulin did not interact with the phospholipids by a hydrophobic interaction, but that calcium binding to spin-labeled calmodulin interfered with phosphatidic acid, phosphatidylserine, phosphatidylglycerol and phosphatidylinositol, and some of these phospholipids inactivated calmodulin. Thus the activity of calmodulin may be regulated in part by some phospholipids.

Binding Sites↗
PubMed1994

Active site structure and stability of the thiol protease papain studied by electron paramagnetic resonance employing a methanethiosulfonate spin label.

The electron paramagnetic resonance (EPR) spin labeling technique has been employed to study the properties and conformation of the thiol protease papain in solution, using (1-oxyl-2,2,5,5-tetramethyl-delta 3-pyrroline-3-methyl) methanethiosulfonate (MTS) as the spin label. The measurements of papain's amidase activity corroborate the EPR results. The major findings are: (i) the motion of the MTS spin label is very sensitive to the active site conformation of papain, which may reflect the location of the pyrroline ring of the spin label near the narrow portion of the active site cleft of papain, and thus there may be intimate interactions between the spin label and its environment; (ii) the active site cleft of papain may have a more open structure at intermediate pH (pH 4.2 to 8.0) than at higher (pH > 8.0) or lower (pH < 4.2) pH, which is consistent with the bell-shape pH curve of the enzyme's amidase activity with the optimum pH at pH 7.00; and (iii) the motion of spin label at the active site of free papain in solution becomes slower upon addition of a denaturant (urea or guanidine hydrochloride), suggesting that the denatured enzyme may have a more closed active site cleft. Urea is more effective than guanidine hydrochloride in denaturing papain at low concentration. However, both urea and guanidine hydrochloride can completely inactivate papain at high concentrations. When an appropriate spin label is selected to label the active site of papain (such as MTS spin label), the EPR spin labeling technique may offer additional insight into the conformation of papain over that obtained by optical methods. These results are discussed in terms of possible studies of biofunctional membranes, opaque assemblies in which a biological molecule is attached to a polymeric membrane.

Binding Sites↗
PubMed1981

Protein synthesis and competitive ESR binding studies with E. coli ribosomes and spin-labeled polynucleotides.

Enzymatically prepared spin labeled copolymers of (U)n were tested for their ability to direct polyphenylalanine synthesis in vitro using E. coli B enzymes and ribosomes. Spin labeling of the C5 position using (RUGT,U)n (1:100) or (RUTT,U)n (1:100) did not alter the amount of polyphenylalanine formed in comparison to (U)n. In contrast, the C4 spin labeled copolymer (ls4U,U)n (1:100) reduced phenylalanine incorporation by 70-75% of the (U)n control levels. ESR monitoring of competitive ribosome binding to equimolar mixtures of polynucleotides was demonstrated with the macromolecular probe (DUTT,dT)n (1:100), the DNA analogue of (RUTT,U)n. The ESR competition approach showed that the affinity of the ribosomes was essentially the same for (dT)n, (A,U,G)n, and (A,U,G)n + tRNArmet.

Bacterial Proteins↗
PubMed1977

Temperature-induced changes in lecithin model membranes detected by novel covalent spin-labelled phospholipids.

Several spin-labelled phospholipids carrying covalently bound 5-doxylstearic acid (2-(3-carboxydecyl)-2-hexyl-4,4-dimethyl-3-oxazolidinoxyl) were intercalated in liposomes of saturated and unsaturated lecithins. Temperature-induced changes of these liposomes, detected by the spin-labelled phospholipids, were found to be in agreement with the previously described transitions of hydrocarbon chains of host lecithins detected by different probes and different techniques, establishing that spin-labelled phosopholipids are sensitive probes for the detection of temperature-induced changes in lecithin model membranes. In addition to the detection of already-known transitions in lecithin liposomes, the coexistence of two distinctly different enviroments was observed above the characteristic transition temperature. This phenomenon was tentatively attributed to the influence of the lecithin polar group on the fluidity of fatty acyl chains near the polar group. Combined with other results from the literature, the coexistence of two environments could be associated with the coexistence of two conformational isomers of lecithin, differing in the orientation of the polar head group with respect to the plane of bilayer. These findings have been discussed in view of the present state of knowledge regarding temperature-induced changes in model membranes.

Animals↗
PubMed1990

The contact sites of sickle hemoglobin as seen by spin probe-spin label techniques.

The spin-labeled tryptophan (TrpSL) was used as a structural probe of hemoglobin (Hb) contact sites. The electron paramagnetic resonance spectral data indicated that the probe exhibits weak binding to Hb with a dissociation constant of 3.2 x 10(-5) and 4.0 mol bound per Hb tetramer. The spectrum suggested that the bound tryptophan was 'partially immobilized' with a correlation time reflecting the environment of the tryptophan binding site of 8.5 s. The topology of the contact sites was investigated by using a dual spin label methodology in which TrpSL and 2H-15N covalently bound to B 93 cysteine residue were used. The electron spin resonance spectral data suggested that the tryptophan binding sites were located within 8-10 A of the nitroxide free radical of spin-labeled Hb. The environment of the contact sites is discussed.

Anemia, Sickle Cell↗
PubMed2005

Membrane-bound orientation and position of the synaptotagmin C2B domain determined by site-directed spin labeling.

Site-directed spin labeling is used to determine the orientation and depth of insertion of the second C2 domain from synaptotagmin I (C2B) into membrane vesicles composed of phosphatidylcholine (PC) and phosphatidylserine (PS). EPR line shapes of spin-labeled mutants located with the Ca(2+)-binding loops of C2B broaden in the presence of Ca(2+) and PC/PS vesicles, indicating that these loops undergo a Ca(2+)-dependent insertion into the membrane interface. Power saturation of the EPR spectra provides a position for each spin-labeled site along the bilayer normal, and these EPR-derived distance constraints, along with a high-resolution structure of the C2B domain, are used to generate a model for the domain orientation and position at the membrane interface. Our data show that the isolated C2B domain from synaptotagmin I penetrates PC/PS membranes, and that the backbone of Ca(2+)-binding loops 1 and 3 is inserted below the level of a plane defined by the lipid phosphates. The side chains of several loop residues are within the bilayer interior, and both Ca(2+)-binding sites are positioned near a plane defined by the lipid phosphates. A Tb(3+)-based fluorescence assay is used to compare the membrane affinity of the C2B domain to that of the first synaptotagmin C2 domain (C2A). Both C2A and C2B bind PC/PS (75:25) membrane vesicles with a micromolar lipid affinity in the presence of metal ion. These results indicate that C2A and C2B have a similar membrane affinity and position when bound to PC/PS (75:25) membrane vesicles. EPR spectroscopy indicates that the C2B domain has different interactions with PC/PS membranes containing 1 mol % phosphatidylinositol 4,5-bisphosphate.

Animals↗
PubMed1975

Bovine serum albumin. Study of the fatty acid and steroid binding sites using spin-labeled lipids.

Three spin-labeled derivatives of stearic acid and two derivatives of palmitic acid have been used to study the structure of the strong fatty acid binding site of bovine serum albumin. The steroid and indole binding sites have been studied using spin-labeled derivatives of androstol and indole, respectively. Paramagnetic resonance and fluorescence quenching data suggest that the fatty acid, steroid, and indole binding sites may be identical. The mobility of the nitroxyl group at C-8 of palmitic acid bound to albumin at a 1:1 molar ratio is unaffected when the carboxyl group is esterified. When the nitroxyl group is located at C-5 on this acid its motion is detectably increased by esterification of the carboxyl group but the magnitude of this change is small. This result suggests that the carboxyl group may play a minor role in the binding of fatty acids to the strongest fatty acid binding site of albumin. When stearic acid derivatives bearing the nitroxide at C-5, C-12, and C-16 are bound to albumin at a ligand to albumin ratio of 1, the order of mobility at 0-30 degrees is C-16 greater than C-12 congruent to C-5. Although motion at the methyl terminus is always greater than at the COOH terminus in the range 0-60 degrees, a simple monotonic increase in chain motion between the two termini is not observed. Arrhenius plots of the motion parameters for these bound fatty acids show two abrupt changes in slope. The temperature ranges for these changes are 15-23 degrees and 38-45 degrees. These results suggest that when one mole of spin-labeled fatty acid is bound to albumin, the protein undergoes a conformational change in each of these temperature ranges.

Animals↗
PubMed2003

Monitoring RNA base structure and dynamics using site-directed spin labeling.

Site-directed spin labeling utilizes site-specific attachment of a stable nitroxide radical to probe the structure and dynamics of macromolecules. In the present study, a 4-thiouridine base is introduced at each of six different positions in a 23-nucleotide RNA molecule. The 4-thiouridine derivatives were subsequently modified with one of three methanethiosulfonate nitroxide reagents to introduce a spin label at specific sites. The electron paramagnetic resonance spectra of the labeled RNAs were analyzed in terms of nitroxide motion and the RNA solution structure. At a base-paired site in the RNA helix, where the nitroxide has weak or no local interactions, motion of the nitroxide is apparently dominated by rotation about bonds within the probe. The motion is similar to that found for a structurally related probe on helical sites in proteins, suggesting a similar mode of motion. At other sites that are hydrogen bonded and stacked within the helix, local interactions within the RNA molecule modulate the nitroxide motion in a manner consistent with expectations based on the known structure. For a base that is not structurally constrained, the mobility is higher than at any other site, presumably due to motion of the base itself. These results demonstrate the general utility of the 4-thiouridine/methanethiosulfonate coupling method to introduce nitroxide spin labels into RNA and the ability of the resulting label to probe local structure and dynamics.

Base Sequence↗
PubMed2002

Structure and dynamics of the beta-barrel of the membrane transporter BtuB by site-directed spin labeling.

Site-directed spin labeling and EPR spectroscopy were used to map two consecutive beta-strands of the putative transmembrane beta-barrel of BtuB. For these studies, a series of 29 consecutive single cysteine mutants of BtuB were produced covering residues 148-176. The proteins were then expressed, reacted with a sulfhydryl-specific spin label, purified in octyl glucoside (OG), and reconstituted into palmitoyloleoylphosphatidylcholine (POPC) bilayers. The labeled residues spanned from the extracellular region (position 148) to the small periplasmic loop (positions 160-163) and back up to the extracellular side (position 176) of BtuB. Continuous wave power saturation in the presence of oxygen or NiAA yielded an i, i + 2 periodicity for the collision frequencies at these sites and demonstrated the presence of a beta-strand structural motif. For both strands studied, the even-numbered residues were found to be exposed to the hydrophobic phase of the bilayer, whereas the odd-numbered residues pointed toward the interior of the barrel and the core of the protein. In addition, the collision parameters yielded the position of the protein within the bilayer. The phase relationship between the oxygen and metal collision frequencies along with the corresponding membrane depth parameters, Phi, indicates that segments 151-159 and 164-172 are within the bilayer. In POPC bilayers, there is a mobility gradient for spin labels along the barrel indicating enhanced backbone flexibility toward the periplasmic surface of the barrel. In POPC/OG mixed micelles, the even-numbered residues facing the hydrocarbon show an increased mobility compared with the bilayer environment whereas the inward-facing side chains show little change in motion. The data indicate that the protein core remains folded in POPC/OG mixed micelles but that this environment increases the backbone fluctuations of the strands. A model for the beta-barrel of BtuB is presented in part on the basis of these EPR data.

Amino Acid Sequence↗
PubMed1976

Effect of Ca2+ binding on troponin C. Changes in spin label mobility, extrinsic fluorescence, and sulfhydryl reactivity.

The Ca2+ binding component (TnC) of troponin has been selectively labeled with either a spin label, N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl) iodoacetamide, or with a fluorescent probe, S-mercuric-N-dansyl cysteine, presumably at its single cysteine residue (Cys-98) in order to probe the interactions of TnC with divalent metals and with other subunits of troponin. The modified protein has the same Ca2+ binding properties as native TnC (Potter, J. D., and Gergely, J. (1975) J. Biol. Chem. 250, 4628), viz. two Ca2+ binding sites at which Mg2+ appears to compete (Ca2+-Mg2+ sites, KCa = 2 X 10(7) M-1) and two sites at which Mg2+ does not compete (Ca2+-specific sites, KCa = 2 X 10(5) M-1). Either Ca2+ or Mg2+ alters the ESR spectrum of spin-labeled TnC in a manner that indicates a decrease in the mobility of the label, Ca2+ having a slightly greater effect. In systems containing both Ca2+ and Mg2+ the mobility of the spin label is identical with that in systems containing Ca2+ alone. The binding constants for Ca2+ and Mg2+ deduced from ESR spectral changes are 10(7) and 10(3) M-1, respectively, and the apparent affinity for Ca2+ decreases by about an order of magnitude on adding 2 mM Mg2+. Thus, the ESR spectral change is associated with binding of Ca2+ to one or both of the Ca2+-Mg2+ sites. Addition of Ca2+ to the binary complexes of spin-labeled TnC with either troponin T (TnT) or troponin I (TnI) produces greater reduction in the mobility of the spin label than in the case of spin-labeled TnC alone, and in the case of the complex with TnI the affinity for Ca2+ is increased by an order of magnitude. The fluorescence of dansyl (5-dimethylaminonaphthalene-1-sulfonyl)-labeled TnC is enhanced by Ca2+ binding to both high and low affinity sites with apparent binding constants of 2.6 X 10(7) M-1 and 2.9 X 10(5) M-1, respectively, calculated from the transition midpoints. The presence of 2 mM Mg2+, which produces no effect on dansyl fluorescence itself, in contrast to its effect on the spin label, shifts the high affinity constant to 2 X 10(6) M-1. Spectral changes produced by Ca2+ binding to the TnC-TnI complex furnish evidence that the affinity of TnC for Ca2+ is increased in the complex. The reactivity of Cys-98 to the labels and to 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2) is decreased by Ca2+ or Mg2+ both with native TnC and in 6 M urea. The reaction rate between Cys-98 and Nbs2 decreases to one-half the maximal value at a Ca2+ concentration that suggests binding to the Ca2+-Mg2+ sites. Formation of a binary complex between TnI and TnC reduces the rate of reaction, and there is a further reduction by Ca2+. The effect of Ca2+ takes place at concentrations that are 1 order of magnitude lower than in the case of TnC alone. These results suggest that the Ca2+ binding site adjacent to Cys-98 is one of the Ca2+-Mg2+ binding sites.

Binding Sites↗
PubMed1986

Interaction of a spin-labeled phenylalanine analogue with normal and sickle hemoglobins: detection of site-specific interactions through spin-label-induced 1H NMR relaxation.

In a preliminary report, we have previously shown that N-[(2,2,5,5-tetramethyl-1-oxypyrrolidin-3-yl)carbonyl]-L-phenyl ala nine tert-butyl ester (SL-Phe) exhibits specific binding to hemoglobin and an antiaggregation activity more than 2 orders of magnitude greater than that of phenylalanine [Lu, H.-Z., Currie, B. L., & Johnson, M. E. (1984) FEBS Lett. 173, 259-263]. Transverse 1H NMR relaxation measurements have been used to investigate the interaction of SL-Phe with hemoglobin molecules by use of the resonances assigned to the C2 protons of the beta 2 His, the beta 143 His, and the beta 146 or beta 97 His residues as intrinsic probes. Distance calculations using the paramagnetically induced relaxation data suggest that the SL-Phe binding site is approximately 12-16 A away from the C2 protons of the beta 2 His and the beta 146 or beta 97 His residues in the (carbonmonoxy)hemoglobin tetramer; for deoxyhemoglobin, the distances are approximately 14-17 A between the SL-Phe binding site and the C2 protons of the beta 2 His, the beta 143 His, and the beta 146 His residues. Calculations using the (carbonmonoxy)hemoglobin crystal atomic coordinates only restrict the probable SL-Phe binding region to the full F and H helices of the beta-chain and a small section of the alpha-chain. For deoxyhemoglobin, the distance calculations provide greater restrictions on the probable binding region, limiting it to small sections of the beta-chain F, G, and H helices near the EF bend and to a few residues on the alpha-chain.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1995

Behavior of cholesterol and spin-labeled cholestane in model bile systems studied by electron spin resonance and synchrotron x-ray.

The behavior of mixed bile salt micelles consisting of sodium taurocholate, egg phosphatidylcholine, and cholesterol has been studied by ESR spin labeling and synchrotron x-ray scattering. Consistent with published phase diagrams, pure and mixed bile salt micelles have a limited capacity to incorporate and, hence, solubilize cholesterol. Excess cholesterol crystallizes out, a process that is readily detected both by ESR spin labeling using 3-doxyl-5 alpha-cholestane as a probe for cholesterol and synchrotron x-ray scattering. Both methods yield entirely consistent results. The crystallization of cholesterol from mixed bile salt micelles is indicated by the appearance of a magnetically dilute powder spectrum that is readily detected by visual inspection of the ESR spectra. Both the absence of Heissenberg spin exchange and the observation of a magnetically dilute powder spectrum provide evidence for the spin label co-crystallizing with cholesterol. In mixed bile salt micelles containing egg phosphatidylcholine, the solubility of cholesterol is increased as detected by both methods. With increasing content of phosphatidylcholine and increasing mole ratio cholesterol/phosphatidylcholine, the anisotropy of motion of the spin probe increases. The spin label 3-doxyl-5 alpha-cholestane is a useful substitute for cholesterol provided that it is used in dilute mixtures with excess cholesterol: the cholesterol/spin label mole ratio in these mixtures should be greater than 100. Despite the structural similarity between the two compounds, there are still significant differences in their physico-chemical properties. These differences come to the fore when cholesterol is totally replaced by the spin-label: 3-doxyl-5a-cholestane is significantly less soluble in bile salt and mixed bile salt micelles than cholesterol and, in contrast with cholesterol, it interacts only very weakly, if at all,with phosphatidylcholine. The potential of the ESR method for detecting cholesterol crystal growth in human bile is discussed.

Bile Acids and Salts↗