Interactions of the cholesterol side-chain with egg lecithin. A spin label study.

The effect in egg lecithin liposomes of cholesterol and cholesterol analogues with side-chains of reduced length on the order parameters of two steroid spin labels has been studied. Analogues with side-chains shorter than cholesterol by more than three carbon cause significantly less ordering than cholesterol. Liposomes containing a cholesterol analogue in which the side-chain is absent cause very little increase in the ordering of a new sterol spin label in which the nitroxide is incorporated into the side-chain. The results suggest that the sterol side-chain exerts a great influence on membrane rigidity within its immediate environment.     

A spin label study of the effects of sterols on egg lecithin bilayers

Paramagnetic resonance of cholestane and three fatty acid probes is used to measure the effects of the addition of cholesterol, 7-dehydrocholesterol and ergosterol to egg phosphatidylcholine bilayers. At low concentrations we find that all three sterols effectively align the bilayers. However, concentrations of ergosterol above 15 mol% disorder and disrupt the bilayers. The observed behavior is explained in terms of a steric model in which the steroid nucleus organizes the bilayer and the bulky egosterol tail disorganizes the bilayer. The three fatty acid spin labels are used to probe the layers at different depths, and the data observed are in agreement with the normal presented.     

A spin label study of horseradish peroxidase.

The topography of the active sites of native horseradish peroxidase and manganic horseradish peroxidase has been studied with the aid of a spin-labeled analog of benzhydroxamic acid (N-(1-oxyl-2,2,5,5-tetramethylpyrroline-3-carboxy)-p-aminobenzhydroxamic acid). The optical spectra of complexes between the spin-labeled analog of benzhydroxamic acid and Fe3+ or Mn3+ horseradish peroxidase resembled the spectra of the corresponding enzyme complexes with benzhydroxamic acid. Electron spin resonance (ESR) measurement indicated that at pH 7 the nitroxide moiety of the spin-labeled analog of benzhydroxamic acid became strongly immobilized when this label bound to either ferric or manganic horseradish peroxidase. The titration of horseradish peroxidase with the spin-labeled analog of benzhydroxamic acid revealed a single binding site with association constant Ka approximately 4.7 . 10(5) M-1. Since the interaction of ligands (e.g. F-, CN-) and H2O2 with horseradish peroxidase was found to displace the spin label, it was concluded that the spin label did not indeed bind to the active site of horseradish peroxidase. At alkaline pH values, the high spin iron of native horseradish peroxidase is converted to the low spin form and the binding of the spin-labeled analog of benzhydroxamic acid to horseradish peroxidase is completely inhibited. From the changes in the concentration of both bound and free spin label with pH, the pK value of the acid-alkali transition of horseradish peroxidase was found to be 10.5. The 2Tm value of the bound spin label varied inversely with temperature, reaching a value of 68.25 G at 0 degree C and 46.5 G at 52 degrees C. The dipolar interaction between the iron atom and the free radical accounted for a 12% decrease in the ESR signal intensity of the spin label bound to horseradish peroxidase. From this finding, the minimum distance between the iron atom and nitroxide group and hence a lower limit to the depth of the heme pocket of horseradish peroxidase was estimated to be 22 A.     

Association-dissociation of histone oligomers. A spin label study

The spin label method has been used to obtain information about conformational changes of histone oligomers taking advantage of the fact that at a low ionic strength and in the presence of other histones about 45% of cysteine residues of histone H3 react with the 3-maleimido-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl spin label. For the labeled complexes H3-H4 and H nu the degree of immobilization of the spin label is a function of the ionic strength. This variation is identical for both complexes within a long range of ionic strengths, including the interval of 0.8-2 M NaCl, under which conditions interactions are known to exist between the tetramer (H3)2 (H4)2 and the dimer (H2A) (H2B). This finding suggests a negligible influence of the dimer for modifying the cysteine residue environment of histone H3 on octamer formation. GuHCl treatment at high ionic strength of the labeled complexes gives rise to a non-lineal increase in the degree of mobility of the spin label. This increase, at low GuHCl concentration (0-0.5 M GuHCl), is interpreted as showing a lowering in rigidity for the Cys residue environment, without affecting the general stability of the tetramer (H3)2 (H4)2. At higher GuHCl concentration (2-3 M GuHCl) the increase in the spin label mobility is related to a dissociation of the complexes in single histones. Our results are consistent with the view that the overall structure of the tetramer, as well as its conformational changes during complex structuration or denaturation, are not strongly affected by the presence of the dimer (H2A) (H2B).     

Interactions of the cholesterol side-chain with egg lecithin a spin label study

The effect in egg lecithin liposomes of cholesterol and cholesterol analogues with side-chains of reduced length on the order parameters of two steroid spin labels has been studied. Analogoues with side-chains shorter than cholesterol by more than three carbons cause significantly less ordering than cholesterol. Liposomes containing a cholesterol analogue in which the side-chain is absent cause very little increase in the ordering of a new sterol spin label in which the nitroxide is incorporated into the side-chain. The results suggest that the sterol side-chain exerts a great influence on membrane rigidity within its immediate environment.     

Structural investigations of DNA-histone complexes. A spin label study.

We have prepared two acridine spin labels, 6-chloro-9-[4-(2,2,6,6-tetramethyl-1-piperidinyloxy)amino]-2-methoxyacridine (I) and 9-[4-(2,2,6,6-tetramethyl-1-piperidinyloxy)amino]-acridine (II) and have used them to study the binding of lysine-rich histone (H1) to DNA using electron spin resonance (ESR). ESR spectra of I in the presence of DNA, polydA-polydT and polydG-polydC were characteristic of highly immobilized radicals with maximum hyperfine splitting (2T11) of 59G, 62.5G and 59G respectively. However, the 2T11 values for II in the same systems were 55.5G, 55.5G and 62.5G respectively. Addition of H1 at a low P/D released ionically bound I and II from DNA. In the presence of 0.1 M NaCl, which prevents ionic binding, H1 still caused a significant release of bound II but not I from DNA. At a high P/D (with or without NaCl) H1 caused no displacement of either I or II. Our findings suggest that H1 does not affect the intercalating sites and probably binds to one of the grooves of DNA, most probably the major groove, and specifically in the A-T-rich regions.     

A spin label study of E. coli membrane vesicles

The phase transition in E. coli membrane vesicles has been investigated by the spin labeling technique. N-oxyl-4′,4′-dimethyloxazolidine derivatives of stearic acid were incorporated into the vesicles. The results suggest that there are two phase transitions in these bacterial membrane vesicles (one at ≈20°C and the other at ≈30°C). These two phase transitions may be related to some of the functional properties of the membranes.     

A spin label study of purple membranes from Halobacterium halobium.

Mammary tumors induced in Sprague-Dawley Rats by the carcinogen 7,12-dimethylbenz(a)anthracene contain a DNA polymerase similar to that found in RNA tumor viruses. It has a molecular weight of 105,000 daltons and is active on the synthetic templates poly(rA):oligo(dT) and poly(rC):-oligo(dG) but is inactive on poly(dA):oligo(dT). This polymerase may be purified more than 300 fold with a 25% yield by ammonium sulfate precipitation, phosphocellulose chromatography and hydroxyapatite chromatography. A similar polymerase is also found in lactating normal rat mammary tissues.     

Phase transitions in sphingomyelin thin filsm. A spin label study

3-Spiro-(2′-(N-oxyl-4′,4′-dimethyloxazolidine)) — cholestane, (I) and 12-spiro-(2′-(N-oxyl-4′,4′-dimethyloxazolidine))-stearic acid (II) have been used as molecular probes to study the interaction of sphingomyelin and cholesterol in both dry and hydrated oriented films at different temperatures. The presence of 50 mole percent cholesterol causes a gel to liquid crystalline phase transition of bovine brain sphingomyelin at 20°C. A temperature induced phase transition involving the phospholipid polar groups has been detected. The mean transition temperature from a rigid to a fluid bilayer lattice structure is 32°C ±0.5°C in hydrated equimolar sphingomyelin — cholesterol films.     

Effect of cholesterol on human erythrocyte membrane. A spin label study

The effect of cholesterol on the membrane fluidity of human erythrocytes has been studied by electron spin resonance (ESR) spectroscopy, sensing the motion of androstane and fatty acid spin labeles in the cell membrane and in vesicles made from extracted phospholipids. 1. Androstane spin label (ASL) was incorporated from ASL-containing phospholipid vesicles into the erythrocyte membrane, essentially by a partition mechanism in proportion to their phospholipid contents. 2. On increasing the cholesterol or ASl content in the cell membrane, the spin label was gradually immobilized. 3. ASL motion in the cell membrane seemed to be primarily determined by the cholesterol/phospholipid molar ratio, regardless of the membrane protein-lipid interaction, as judged from the temperature effects on the ESR spectra of both membranes. 4. However, glutaraldehyde pretreatment induced considerable changes of the cholesterol-lipid interaction in the cell membrane, i.e., strong immobilization and cluster formation of ASL were observed.     

A spin label study of lipid oxidation catalyzed by heme proteins.

Rapid loss of the electron spin resonance signal from a variety of spin labels is observed when ferricytochrome c or metmyoglobin are combined with lipids. Evidence is presented that this loss of signal can be used as a sensitive method to study lipid oxidation catalyzed by heme proteins. Under aerobic conditions and with lipids which bind the heme protein, the kinetics of the oxidation process as observed by the spin label method are identical to the kinetics previously observed by measurements of oxygen uptake. Use of pre-oxidized lipids under anaerobic conditions indicates that cytochrome c reacts with a product of lipid oxidation. Kinetic studies of the anaerobic reaction indicate that cytochrome c reacts rapidly with lipid oxidation products in membrane areas far larger than the area occupied by cytochrome c, implying rapid transport of reactive species within the membrane interior in directions parallel to the membrane surface. Under anaerobic conditions, reaction of cytochrome c with lipid oxidation products appears to produce a relatively long lived (hours) species located in the hydrophobic portion of the membrane, which is capable of subsequent reaction with lipid-soluble spin labels.     

A spin label study of immobilized enzyme spectral subpopulations

Electron spin resonance (ESR) spin label studies have been carried out to examine the active site conformation of alpha-chymotrypsin before and after immobilization on two types of organic polymer supports: Amberlite XAD-8 and XAD-2. alpha-Chymotryspin was first chemically modified by reaction with methyl-4-phenylbutyrimidate and then inhibited by the active site spin label 4-(2,2,6,6-tetramethyl-piperdine-1-oxyl)-m-flurosulfonylbenzamide. In general, the ESR spectra of the active site lable revealed no significant changes in conformation for most of the enzyme before or after derivatization. On the other hand, two spectral subpopulations (A and B) of spin-labeled enzyme were characterized on the basis of their ESR spectra after immobilization on Amberlite XAD-8. Spectral subpopulation A (distinguished by a highly restrained spectrum) appeared to retain its active site structure and conformation and represented a large majority of the labeled chymotrypsin on the beads. Its presence correlated with the high activity and stability of phenylbutyramidinated chymotryspin on the Amberlite XAD-8 beads. Spectral subpopulation B (distinguished by a very weakly constrained spectrum) appeared to reflect loosely bound or denatured enzyme which was removable upon washing with 40% (v/v) ethylene glycol. Two methods for examining solvent accessibility to the active site lable of the kinetics of ascorbate reduction suggested that both spectral subpopulations had identical accessibilities to the bulk solvent. Paramagnetic broadening of the signal by K(3)Fe(CN)(6) revealed differences in the spin-spin broadening of the A and B components but is deemed and inappropriate indicator of solvent accessibility.     

Bacteriorhodopsin,boundary lipid and protein conformers. A spin label study

A spin label study, as a function of temperature, has been made with the bacteriorhodopsin membrane using a stearic acid spin label. The ESR spectra show a strong variation with temperature and the presence of isosbestic points. The spectra are interpreted as indicating the presence of a two-component system with an activation energy (approx. 14 kcal/mol) corresponding to a protein conformational change. This activation energy is similar to that deduced from recent flash photolysis studies.It is concluded that the spin label is sensitive to the temperature-dependent protein conformational change in this membrane system.     

A spin label study on human low density lipoprotein

Selective labeling with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)-maleimide of human serum LDL has been performed. The spin-labeled LDL exhibited an ESR spectrum containing signals of a strongly immobilized component only. The signals were completely reversible between 4 degrees C and 37 degrees C and fairly stable at each temperature. The spin-labeled LDL which was prepared by the usual method exhibited an ESR spectrum containing signals of both strongly immobilized and weakly immobilized components (5, 6). The latter was unstable above 25 degrees C and changed irreversibly. The strongly binding site showed higher affinity for the nitroxide radical than the weakly binding site, and two kinds of the strongly binding site were demonstrated kinetically. The rate of binding of the nitroxide radical to the two kinds of strongly binding site were estimated to be 4.7 x 10(4) M-1 . day-1 and 0.16 x 10(4) M-1 . day-1 at pH 7.4 and 4 degrees C, respectively. Both the strongly immobilized and weakly immobilized radicals were reduced with ascorbate at the same rate. It was also shown on gel filtration of the SDS-treated LDL derivatives that the strongly immobilized component was on the apoprotein B moiety, whereas either noncovalent binding to LDL or binding to some small molecular species other than protein was suggested for the weakly immobilized component.     

Steroid modulation of human serum albumin binding properties. A spin label study.

The binding isotherm and unique electron spin resonance spectral characteristics of a monoanionic spin label (1-gamma-aminobutyrate-5-N-(1-oxyl-2,2,6,6-tetramethyl-4-aminopiperidinyl)-2,4-dinitrobenzene) and a dianionic spin label (1-glutamate-5-N-(1-oxyl-2,2,6,6-tetramethyl-4-aminopiperidinyl)-2,4-dinitrobenzene) are used to prove the steroid modulation of serum albumin binding properties. Effects of a selected number of steroids (progesterone, testosterone, estradiol, aldosterone, estriol, corticosterone, deoxycorticosterone, hydrocortisone, and cortisone) on the binding isotherm of the monoanionic spin label binding to serum albumin have been determined. At the steroid/albumin ratio of 0.5 to 1, progesterone, testosterone, and estradiol enhance binding of the spin label at all concentrations studied. However, the remaining steroids exert an inhibitory effect at low spin label/albumin ratios and an enhancement effect at high spin label/albumin ratios. Progesterone and cortisone effects on the resonance spectra of the spin label bound to serum albumin confirm the enhancement and displacement properties of these ligands. Thus, like fatty acids, steroids may bind to either the primary or secondary bilirubin binding sites and also allosterically perturb the binding properties of serum albumin. The in vivo importance of the steroid-albumin interaction is discussed.     

A spin label study of conformational changes in cytochrome c

Spin-labeled pig heart cytochromes c singly modified at Met-65, Tyr-74 and at one of the lysine residues, Lys-72 or Lys-73, were investigated by the ESR method under conditions of different ligand and redox states of the heme and at various pH values. Replacement of Met-80 by the external ligand, cyanide, was shown to produce a sharp increase in the mobility of all the three bound labels while reduction of the spin-labeled ferricytochromes c did not cause any marked changes in their ESR spectra. In the pH range 6-13, two conformational transitions in ferricytochrome c were observed which preceded its alkaline denaturation: the first with pK 9.3 registered by the spin label at the Met-65 position, and the second with pK 11.1 registered by the labels bound to Tyr-74 and Lys-72(73). The conformational changes in the 'left-hand part' of ferricytochrome c are most probably induced in both cases by the exchange of internal protein ligands at the sixth coordination site of the heme.