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.     

A spin label study of egg white avidin.

Avidin is a tetrametric protein (mass 68,000 daltons) that binds 4 molecules of vitamin biotin (1). The biotin binding sites, 1 per subunit, are grouped in two pairs at opposite ends of the avidin molecule (GREEN, N.M., KONIECZNY, L., TOMS, E.J., and VALENTINE, R.C. (1971) Biochem. J. 125, 781). We have studied the topography of the avidin binding sites with the aid of four spin-labeled analogs of biotin: 4-biotinamido-2,2,6,6-tetramethyl-1-piperidinyloxy (II), 3-biotinamido-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (III), 3-biotinamidomethyl-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (IV), 4-(biotinylglycyl)-amino-2,2,6,6-tetramethyl-1-piperidinyloxy (V). Fluorescence and optical absorption spectroscopy indicated that II to V occupied the same binding sites on avidin as did biotin. The electron spin resonance spectrum of the 4:1 complex between II and avidin contained broad line components characteristic of a highly immobilized spin label. Dipole-dipole interactions between spin labels bound to adjacent sites split each of the three major hyperfine lines into doublets with a separation of 13.8 G. The distance between adjacent bound nitroxide groups was calculated from this splitting to be 16 A. The dissociation of the 4:1 complex between II and avidin was biphasic with approximately half of the labels dissociating at a rate (kdiss equal to 2.51 times 10- minus 4 s- minus 1) that was much faster than the remainder (kdiss equal to 1.22 times 10- minus 5 s- minus 1). The electron spin resonance spectrum of the 2:1 complex between II and avidin clearly showed that, immediately after mixing, the spin labels were distributed in a random fashion among the available binding sites but that they slowly redistributed themselves so that each label bound to a site which was adjacent to an unoccupied site. The final time-independent electron spin resonance spectrum exhibited a splitting 69 G between the low and high field hyperfine lines which is characteristic of a highly immobilized, noninteracting spin label. Spin labels III and IV interacted with avidin in a similar fashion to that described for II with the exception that their dipolar splittings were 11.9 G and 14.2 G, respectively. From these splittings it was estimated that the distance between adjacent avidin-bound nitroxides was 16.7 A for labeled III and 15.7 A for label IV. The electron spin resonance spectrum of label V bound to avidin was characteristic of a noninteracting highly immobilized nitroxide with a maximum splitting of 62 G. The spectrum of V bound to avidin was independent of both time and the amount of bound label. The rate of dissociation of V from a 4:1 complex with avidin was monophasic. A model is proposed in which the recognition site for the heterocyclic ring system of biotin is represented as a cleft located within a hydrophobic depression in the surface of avidin.     

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.     

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 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.     

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.     

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 the urinary bladder luminal membrane

Spin label experiments have been carried out on the urinary bladder luminal membrane of the bovine transitional epithelium employing the 5-, 7-, 12-, and 16-doxyl substituted stearic acid methyl esters, and compared for reference to similarly labeled bovine erythrocytes. The bladder membranes are significantly different from the bovine red blood cell membranes and show a lower order and polarity near the membrane surface. This fact and the general similarity of results for the bladder and isolated plaque membranes suggests that the highly organized proteins of the bladder membrane may act as a coat on the lipid bilayer and, while intrinsic in nature, do not significantly perturb the hydrophobic core of the lipid bilayer.     

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.     

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.     

ESR spin label studies of the nucleosome core particle and histone core

An imidazole spin label has been used to study the accessibility and conformational state of tyrosines in both the nucleosome core particles and histone core extracted from chicken erythrocytes. About 40% of the tyrosyl residues in the histone core can be labeled under nondenaturing conditions. However, less than 15% of the tryosyls in the nucleosome core particle can be labeled even at 200- to 300-fold M excess of label. The effect of urea on the conformational state of the spin-labeled tyrosyls in both the nuclesome core particles and the histone core has been studied. Ionic effects on the spin-labeled nucleosome core have been investigated. Several conformational transitions are observed in the range of 1 mM NaCl to 2.5 M NaCl. Three major transitions are found at 0.1 M to 0.6 M, 0.7 M to 1.8 M and 2 M to 2.5 M NaCl, respectively. The observed changes can be interpreted as swelling and conformational change of the inner histone core, gradual separation of DNA from the histone core, and tightening of the histone core.     

Fatty acid enhancement of human serum albumin binding properties. A spin label study.

The introduction of a new spin-labeled anionic ligand, 1-gamma-aminobutyrate-5-N-(1-oxyl-2,2,6,6-tetramethyl-4-aminopiperidinyl)-2,4-dinitrobenzene, is reported. Under the experimental conditions, the first molar equivalent of this ligand is 93% bound to human serum albumin. With the addition of palmitate, the free spin label concentration decreases greatly, by almost 80%, in the presence of a fatty acid:albumin ratio of 3:1 to 4:1. The spectral characteristics of the bound spin label are also affected. The changes seen in the intensity of and the splitting between the high and low field extrema are indicative of perturbations of the protein molecule. It is seen then that the binding of each molar equivalent of fatty acid effects the conformation state of albumin and allosterically affects albumin binding properties. Computer spectral subtractions, furthermore, suggest that the binding of the first molar equivalent of palmitate specifically increases the affinity of the first two 1-gamma-amino-butyrate-5-N-(1-oxyl-2,2,6,6-tetramethyl-4-aminopiperidinyl)-2,4-dinitrobenzene binding sites. The present results indicate that fluctuations in serum free fatty acid levels within the physiological range may have a major modulatory effect on the free serum levels of certain drugs and/or physiological substances that bind to albumin.     

A spin label study of erythrocyte membranes during simulation of freezing

Summary Human erythrocytes were labeled with stearic acid spin labels, and no change was detected in membrane fluidity under hyperosmotic stress, going from isotonicity to about 3000 mOsm. Intact erythrocytes labeled with an androstane spin label and submitted to simulation of freezing show the onset of irreversible structural breakdown occurring in a saline solution at 2,000 mOsm. Ghosts labeled with maleimide spin label (4-maleimide-2,2,6,6-tetramethylpiperidinooxyl) when submitted to solutions of increasing osmolalities (pH 7.4), exhibit protein conformational changes that are irreversible after a simulated freeze-thaw cycle. After sonication of maleimide spin-labeled ghosts, membrane buried sulfhydryl groups become exposed. Such preparations showed behavior similar to the unsonicated when in saline hyperosmolal medium (pH 7.4). Such results suggest the ionic strength of the medium as the determining factor of the detected conformational changes. Maleimide spin-labeled ghosts in 300 mOsm saline solution (pH 7.4) were treated with ascorbic acid (spin destruction of nitroxides), and the kinetic analysis indicates that 65% of the labeled sites are located at the external interface of the membrane or in hydrophilic channels. Deformation and rearrangements of membrane components in solutions of increasing osmolalities apparently are related to protein conformational changes, on the outside surface of erythrocyte membranes, with a significant amount being structurally dissociated of lipids.     

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 metmyogloblin 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.     

Fluidity of rat liver Golgi membranes in streptozotocin diabetes. A spin label study

The mobility of 5-doxylstearic acid spin label (5-SASL) in the intact rat liver Golgi membranes of streptozotocin diabetes was studied as a function of free blood sugar level and temperature. During development of diabetes, indicated by the increase of the free blood sugar level, the membrane fluidity measured in the physiological temperature range (1) does not change in comparison with control in light diabetes, (2) decreases significantly in advanced diabetes and (3) again increases to the control level in heavy diabetes (the free blood sugar levels being 200-250 mg/100 ml, 250-350 mg/100 ml and greater than 350 mg/100 ml, respectively). The development of streptozotocin diabetes is accompanied by significant changes in lipid composition of liver Golgi membranes as also shown in our previous observations. The measurements of motion of 5-SASL in Golgi membranes as well as in vesicles, made from commercially available lipids of composition close to the liver Golgi membranes, show that a decrease of cholesterol contents is the main factor which induces the increase membrane fluidity. We suggest that in the heavy diabetes the hemostatic regulation in the lipid composition leads to minimization of alterations in membrane fluidity to obtain comparatively normal activity of certain membrane enzymes.     

Dynamic spin label study of the barstar-barnase complex

The dynamic spin label method was used to study protein-protein interactions in the model complex of the enzyme barnase (Bn) with its inhibitor barstar. The C40A mutant of barstar (Bs) containing a single cysteine residue was modified with two different spin labels varying in length and structure of a flexible linker. Each spin label was selectively bound to the Cys82 residue, located near the Bn-Bs contact site. The formation of the stable protein complex between Bn and spin labeled Bs was accompanied by a substantial restriction of spin label mobility, indicated by remarkable changes in the registered EPR spectra. Order parameter, S, as an estimate of rapid reorientation of spin label relative to protein molecule, was sharply increasing approaching 1. However, the rotational correlation time tau for spin-labeled Bs and its complex with Bn in solution corresponded precisely to their molecular weights. These data indicate that both Bs and its complex with Bn are rigid protein entities. Spin labels attached to Bs in close proximity to an interface of interaction with Bn, regardless of its structure, undergo significant restriction of mobility by the environment of the contact site of the two proteins. The results show that this approach can be used to investigate fusion proteins containing Bn or Bs.