Nonpolar Sites Research Articles

Impermeant potential-sensitive oxonol dyes: I. Evidence for an "on-off" mechanism.

This series of papers addresses the mechanism by which certain impermeant oxonol dyes respond to membrane-potential changes, denoted delta Em. Hemispherical oxidized cholesterol bilayer membranes provided a controlled model membrane system for determining the dependence of the light absorption signal from the dye on parameters such as the wavelength and polarization of the light illuminating the membrane, the structure of the dye, and delta Em. This paper is concerned with the determination and analysis of absorption spectral changes of the dye RGA461 during trains of step changes of Em. The wavelength dependence of the absorption signal is consistent with an "on-off" mechanism in which dye molecules are driven by potential changes between an aqueous region just off the membrane and a relatively nonpolar binding site on the membrane. Polarization data indicate that dye molecules in the membrane site tend to orient with the long axis of the chromophore perpendicular to the surface of the membrane. Experiments with hyperpolarized human red blood cells confirmed that the impermeant oxonols undergo a potential-dependent partition between the membrane and the bathing medium.

Enzymic Formation of β-D-Fructofuranosides from Sucrose: Activity and Selectivity of Inventase in Mixtures of Water and Alcohol

Abstract Invertase-catalysed alcoholysis of sucrose in water - primary alcohol mixtures containing up to 70% organic solvent shows 5-40% alkyl β-D-fructofuranoside formation. No formation of fructosides was observed under anhydrous conditions. The kinetics of the competing reactions of water and primary alcohol as fructosyl acceptors with sucrose as fructosyl donor were studied in order to establish the scope and limitations of the method. On a molar basis, water was a relatively unreactive acceptor. 6-Kestose formation due to sucrose itself as a fructosyl acceptor was suppressed by the presence of aliphatic alcohols. The results have been explained by assuming an nonspecific non-polar binding site to be present in the enzyme cavity near the specific polar binding site for the β-D-fructofuranosyl group of the substrate.

Pyridoxal phosphate site in glycogen phosphorylase b: structure in native enzyme and in three derivatives with modified cofactors.

The detailed environment of the essential cofactor pyridoxal 5'-phosphate in glycogen phosphorylase b, resulting from crystallographic refinement at 1.9-A resolution, is described. The pyridoxal ring is buried in a nonpolar site containing three aromatic rings while the 5'-phosphate group is highly solvated and makes only three direct contacts to the protein. The pyridine nitrogen interacts via a water with protein atoms [main chain carbonyl oxygen (Asn-133) and OH of tyrosine (Tyr-90)]. The crystal structures of three active derivatives of phosphorylase reconstituted with 5'-deoxypyridoxal 5'-methylenephosphonate (PDMP), 6-fluoropyridoxal 5'-phosphate (6-FPLP), and pyridoxal (PL) in place of the natural cofactor have been determined at 2.5-A resolution. The results for PDMP-phosphorylase show a closer proximity of the phosphonate group to the NZ atom of a lysine (Lys-574) than that observed in the native enzyme, consistent with 31P NMR studies that have shown a change in ionization state of the phosphonate group compared to the native cofactor phosphate. The replacement of the polar 5'-ester linkage by a CH2 group results in a small shift of a water and its hydrogen-bonded tyrosine (Tyr-648). In 6-FPLP-phosphorylase the fluorine is accommodated with no significant change in structure. It is suggested that substitution of the electronegative fluorine at the 6-position may result in lower activity of 6-FPLP-phosphorylase through a strengthening of hydrogen-bonded interactions to the pyridine nitrogen N1.(ABSTRACT TRUNCATED AT 250 WORDS)

Design, synthesis, and testing of potential antisickling agents. 10. (2,2-Dimethylchroman-6-yl)alkanoic acids.

Five (2,2-dimethylchroman-6-yl)alkanoic acids were synthesized and tested for antigelling activities. It was envisioned that these agents might bind via hydrophobic bonding to nonpolar sites of the "donor-acceptor" regions of hemoglobin S. Several (2,2-dimethylchroman-6-yl)alkanoic acids containing 1-4 carbon atoms on the side-chain residue were designed to interact at the acceptor site, were synthesized, and were found to be moderately potent antigelling agents. The weak activity observed for two of the acids at low concentrations is rationalized in terms of weak binding affinities or multiple binding to active and nonactive sites. The effect of these compounds on shifting the allosteric equilibrium was small or negligible. The low toxicity of one of the (2,2-dimethylchroman-6-yl)alkanoic acids demonstrates the potential use of yet another class of compounds that can be modified in the development of antisickling agents.

Anesthetic potencies of secondary alcohol enantiomers.

The Meyer-Overton rule has been interpreted to mean that general anesthetics act at a nonpolar site, either in a lipid bilayer or a protein. Optical isomers, also called enantiomers, are pairs of compounds with the same molecular formula and functional groups, but which differ in the arrangement of the groups around an "asymmetric" carbon atom and in the direction they rotate plane-polarized light. By definition, enantiomers that are anesthetics can distinguish between stereoselective and nonselective sites of anesthetic action. We used such enantiomers to determine whether anesthetics are stereoselective in their actions on animals by measuring the potencies of a homologous series of secondary aliphatic alcohols from 2-butanol through 2-octanol in tadpoles, using reversible loss of righting reflex as the endpoint. None of the isomeric pairs exhibited significant differences in potency. Anesthetic potency increased logarithmically with the number of carbon atoms in the hydrocarbon chain of the alcohol. The ED50 +/- SE (mM) for the (+) and (-) forms of the alcohols, respectively, were as follows: 2-butanol 17 +/- 1.2, 17 +/- 1.1; 2-pentanol 4.7 +/- 0.28, 4.8 +/- 0.27; 2-hexanol 1.33 +/- 0.068, 1.42 +/- 0.079; 2-heptanol 0.32 +/- 0.011, 0.33 +/- 0.020; and 2-octanol 0.063 +/- 0.0042, 0.061 +/- 0.0032. These data demonstrate a lack of stereoselectivity in the interactions between the anesthetic secondary alcohols and their site of action in animals.

Theory of distortion-induced I.R. and Raman intensity of forbidden modes of molecular units in crystals

Using the principles of group theory and approximation methods, a quantitative theory of distortion-induced infrared and Raman intensity due to forbidden modes of molecular units at non-polar site symmetries in crystals has been discussed. Among all forbidden modes only those which belong to , attain distortion induced activity (DIA) of first order where α ij/an is a component of anisotropic polarizability tensor; others attain higher order DIA and are hardly expected to be observed in I.R./Raman spectra due to their weak intensity.

Ca 2+ binding and charge movements in membranes of platelets and sarcoplasmic reticulum

The properties of the Ca 2+-pump system of platelet microsomes isolated without Ca 2+-precipitating anions are studied. Passive Ca 2+ binding to the microsomes takes place in a noncooperative manner with K d = 0.7 μM. Half-maximal stimulation of ATP-dependent transport occurs at 0.4 μM Ca 2+. The velocity of Ca 2+ uptake, Ca 2+ capacity and the level of phosphoprotein in platelet microsomes are significantly lower than in cardiac microsomes. Energization of platelet and muscle microsomes and activation of intact platelets result in opposite charge redistribution in hydrophobic regions of the membranes. It is concluded that these charge movements are caused by Ca 2+ binding to and dissociation from nonpolar binding sites in the membranes.

Rotational dynamics of 4-aminobutyrate aminotransferase

The fluorescence dye 1-anilinonaphthalene-8-sulfonate (ANS) was used as a probe of non-polar binding sites in 4-aminobutyrate aminotransferase. ANS binds to a single binding site of the dimeric protein with a K d of 6 μM. Nanosecond emission anisotropy measurements were performed on the ANS-enzyme in an effort to detect independent rotation of the subunits in the native enzyme. The observed rotational correlation time (φ = 65 ns) corresponds to the rotation of a rather rigid dimeric structure. The microenvironment surrounding the natural probe pyridoxal-5-P covalently bound to the dimeric structure was explored using 31P-NMR at 72.86 MHz. In the native enzyme, the pyridoxal-5-P 31P-chemical shift is pH-independent, indicating that the phosphate group is well protected from the solvent. The correlation time determined from the 31P-spectrum of the aminotransferase exceeds the value calculated for the hydrated spherical model (φ = 40 ns). It is concluded that the phosphate of the pyridoxal-5-P molecule is rigidly bound to the active site of 4-aminobutyrate aminotransferase.

Nanosecond spectroscopy of a dimeric enzyme: plasma amine oxidase

The fluorescence dye 1-anilino-naphthalene-8-sulphonic acid (ANS) was used as a probe of non-polar binding sites in the enzyme plasma amine oxidase. Steady fluorescence measurements indicate that ANS binds to a single binding site of the dimeric enzyme with a dissociation constant of 5 microns. This binding site is different from the catalytic binding site. Nanosecond emission anisotropy measurements were performed on the ANS-enzyme in an effort to detect independent rotation of the subunits in the native enzyme. The observed rotational correlation time (phi = 105 ns) corresponds to the rotation of a rigid dimeric macromolecule. A rotational correlation time of 120 ns was obtained with the enzyme labelled with pyrenebutyric acid. It is concluded that the dimeric enzyme does not exhibit any modes of flexibility due to independent rotation of the subunits in the nanosecond range.

Study of steroid-protein interactions by electron spin resonance spectroscopy. Binding of a spin-labelled dihydrotestosterone to bovine serum albumin

The interaction of bovine serum albumin with dihydrotestosterone bearing a spin label at C-3 was studied using electron spin resonance (ESR) spectroscopy. Quantitative binding parameters ( K a≈10 5M t-1 ; maximum binding capacity; two sites/mol albumin) obtained by ESR were in good agreement with those given by equilibrium dialysis. ESR study at various temperatures allowed the calculation of the thermodynamic parameters of the steroid-protein interaction: ΔG = −6.8 kcal/mol; ΔH = −7.9 kcal/mol; ΔS = −3.2 cal/mol per degree and confirmed a transition temperature of about 65°C for albumin. Na, Li and Ca salts had a generally favorable effect on the interaction whereas other ions (e.g. Hg, Cu) impaired the binding process. Study of the width of the ESR spectra of the protein-bound spin-labelled steroid and extrapolation of a 2 T value to infinite viscosity ( A zz coupling constant ) indicated a non-polar binding site, which became increasingly hydrophobic as the temperature was raised. Since this methodology can give both pertinent quantitative and qualitative data, ESR spectroscopy should be of value in the study of steroid-protein interactions of biological significance.

Biochemical studies of taste sensation. III. Preparation of a suspension of bovine taste bud cells and their labeling with a fluorescent probe

A method to prepare suspensions of taste bud cells is described. Bovine circumvallate papillae, which contain most of the taste buds in this animal, are incubated in collagenase-containing medium and the epidermal sidewall tissue is then dissected from the inner gelatinous dermis. The sidewall tissue, which contains the taste buds, is gently homogenized by manual operation of an all-glass homogenizer with a loose-fitting pestle. The suspended material is separated on a discontinous Ficoll gradient (2%, 8%, 10%, 12% w/w). The material banding at the 8-2% interface is greatly enriched in spindle-shaped cells that are morphologically similar to taste bud cells as they appear in situ. These cells are not seen when the procedure is done with tissues devoid of taste buds, namely the upper surface of the circumvallate papilla or epithelium from the intermolar eminence. Fluorescence analysis indicates that the hydrophobic probe, 8-anilino-1-naphthalenesulfonate (ANS), binds to relatively nonpolar sites in the suspension. It is postulated that the probe is adsorbing onto the surface membrane of the cell. These preparations may be useful in studying specificity and transduction in taste sensation.

Mechanisms of lipid peroxidation in erythrocytes of vitamin E-deficient rats and in phospholipid model systems

Erythrocytes from vitamin E-deficient and control rats were peroxidized by glucose oxidase-glucose or dialuric acid. Losses of polyunsaturated fatty acids from membrane phospholipids, and of dimethylacetals from plasmalogens, were quantitated by gas-liquid chromatography. Similar treatment of solubilized or micellar phospholipids or plasmalogens in vitro showed that in both erythrocytes and micellar systems, arachidonic acid and the 16-carbon plasmalogen are most susceptible to peroxidation by either reagent. The same narrow concentration range of dialuric acid found effective in peroxidizing erythrocytes from tocopherol-deficient rats was also found effective in peroxidizing micellar phospholipids in vitro. Partially peroxidized erythrocytes from tocopherol-deficient rats were subjected to treatment with phospholipase A or phospholipase C. Hemolysis by either phospholipase was accelerated in partially peroxidized cells as compared to controls, suggesting that peroxidation exposes both polar and nonpolar lipid sites in the erythrocyte membrane.

The application of nanosecond fluorometry to an allosteric protein

The fluorescent probe 1-anilino-8-naphthalene sulfonate, when bound by the inactive form of phosphorylase b, behaves as if distributed between two different binding sites, whose bound dye emits fluorescence with decay times of 19 and 8 nanosecs. In 0.1 M glycylglycine, pH 7.0, the addition of the allosteric activator adenosine-5′-phosphate or the substrate glucose-1-phosphate results in the progressive loss of the component of longer decay time. At intermediate levels of activator the decay curves could be represented as the sum of the weighted contributions of two species, corresponding to those prevailing in the absence of activator and in excess activator. The activity of glycogen phosphorylase b, which is one of the most extensively studied allosteric enzymes, is controlled by the allosteric activator AMP (1, 2). Binding studies have shown that the binding of AMP in the absence of substrate is strongly cooperative, with a value of the Hill coefficient of 1.7 (2). Binding and kinetic studies have indicated that strong heterotropic cooperativity exists between the combination of the enzyme with AMP and either of the substrates glucose-1-phosphate and inorganic phosphate (1, 2, 3, 4). Phosphorylase b (molecular weight 185,000) consists of two equivalent subunits of molecular weight 92,500. Each subunit contains a single strong binding site for AMP(1, 2, 3). The AMP-induced activation of phosphorylase b has usually been attributed to a conformational transition, AMP being preferentially bound by the active form (1). The high degree of cooperativity of AMP binding has been attributed to the simultaneous conversion of both subunits to the active conformation upon the binding of a molecule of AMP by one subunit (1, 2). The existing kinetic studies have been interpreted as indicating that the binding of substrate likewise favors the transition to the active form, although differences of opinion exist as to the number and equivalence of the active states (1, 2, 5). However, a basic weakness of the model described above is the paucity of direct physical evidence for a conformational change. In recent years a rapid evolution of technique has made it possible to monitor the time decay of fluorescence directly (6). The technique of nanosecond fluorometry has been applied to a number of systems with the objective of analyzing complex decay curves or detecting excited state reactions (7). With the development of programs for analyzing multicomponent decay curves in terms of the lifetimes and amplitudes of the individual emitting species (8) it has become possible to apply nanosecond fluorometry to the problem of monitoring conformational transitions of proteins, and in particular, to the activator - or substrate - induced transformations of allosteric proteins. In general, when emission occurs from a collection of sources with different decay times, we have for the fluorescence intensity i (t) as a function of time, t: (1) i(t) = ∑ j α j, e, -t τ j where α j and τ j are the amplitude and decay time, respectively, corresponding to the jth component. If the number of components is small, the experimental curves of i(t) versus t may, in principle, be analyzed by the method of moments to yield the set of values of α j and τ j. In this report, we shall describe the results of the application of nanosecond fluorometry to the phosphorylase b system, using the fluorescent probe 1-anilino-8-naphthalene sulfonate (ANS). This probe, which is virtually non-fluorescent in aqueous solution, acquires an intense fluorescence when bound by a non-polar site on a protein (9, 10). In an earlier report, Seery and Anderson have found that the fluorescence intensity of ANS bound to phosphorylase b is sharply reduced in the presence of AMP or substrates. From parallel equilibrium dialysis studies, it was concluded that the greater part of the effect arose from the dissociation of ANS, although it was not possible to demonstrate a quantitative correlation (11). For this reason, it was not possible to make any statement as to the quantum yield or excited lifetime of the residual bound dye.

Thermodynamics of Unfolding of Lysozyme in Aqueous Alcohol Solutions

Abstract The thermal denaturation of lysozyme in aqueous alcohol solutions has been investigated through ultraviolet difference spectrophotometry and optical rotation measurements. Alcohols used were CH3OH, C2H5OH, and C3H7OH; concentrations up to 50% (v/v) of alcohol in water were employed. It was found that upon increasing the size or the concentration of the aliphatic alcohol, the denaturation temperature is gradually depressed. At low alcohol concentration, the α helix content of the protein decreases through the thermal transition. However, on increasing alcohol size or concentration, the α helix content goes through a maximum during denaturation. Apparent equilibrium constants and corresponding van't Hoff heats of denaturation were determined. The data indicate that the depression of the denaturation temperature due to addition of alcohols is essentially entropy dependent. It is suggested that these results may be explained in terms of a theoretical expression containing a binding constant for the alcohol to the nonpolar sites of the protein plus a diluent term accounting for changes in the dilution parameters of different conformers.

L-kynurenine: A fluorescent probe of serum albumins

The specific interaction of l-kynurenine with serum albumins was investigated by fluorescence spectroscopy. The addition of l-kynurenine to a solution of serum albumin induces in parallel to fluorescence enhancement a marked increase in the polarization of fluorescence of the ligand. Binding experiments conducted at constant protein concentration and varying ligand concentration reveal that the l-kynurenine—protein complex is characterized by an association constant ( K a ) of 2.8 · 10 5 M −1. l-kynurenine complexed to bovine serum albumin displays a maximum of emission at 435 nm, while l-kynurenine in hydrogen bonding solvents is characterized by a maximum of emission at 460 nm. This blue shift in the band position of the emission spectrum is more pronounced than the corresponding shift in the absorption spectra. The possible relationship of these spectral changes to the binding of l-kynurenine to a non-polar binding site is discussed; it is proposed that during its brief fluorescence lifetime the molecule of l-kynurenine bound to the protein does not interact with the surrounding solvent.

EFFECTS OF GENERAL ANAESTHETICS ON MICROTUBULES

Abstract General anaesthetics affect a number of cellular systems (e.g., cleavage figures in sea-urchin eggs, mitosis in metaphase, the beating of cilia); cytoplasmic microtubules are found in all these situations. These microtubules, which are thought to give rigidity to cytoplasm, are rings of protein units bound longitudinally; individual units can be depolymerised to subunits of molecular weight around 60,000. Several agents (cold, high hydrostatic pressure, and colchicine) can reversibly depolymerise microtubules, probably by interference with protein interactions; heavy water favours polymerisation. Cold, high pressure, or colchicine can produce narcosis, and heavy water can antagonise effects of general anaesthetics. It is suggested that microtubular proteins are also susceptible to reversible depolymerisation by anaesthetics, and that this is achieved by binding to non-polar sites on globular proteins. How this depolymerisation results in narcosis is not yet clear, but one possibility (suggested by the abundance of microtubules in neurons) is that microtubules participate in nerve-impulse transmission.

EFFECTS OF GENERAL ANÆSTHETICS ON MICROTUBULES: A Possible Mechanism of Anaesthesia

General anæsthetics affect a number of cellular systems (e.g., cleavage figures in sea-urchin eggs, mitosis in metaphase, the beating of cilia); cytoplasmic microtubules are found in all these situations. These microtubules, which are thought to give rigidity to cytoplasm, are rings of protein units bound longitudinally; individual units can be depolymerised to subunits of molecular weight around 60,000. Several agents (cold, high hydrostatic pressure, and colchicine) can reversibly depolymerise microtubules, probably by interference with protein interactions; heavy water favours polymerisation. Cold, high pressure, or colchicine can produce narcosis, and heavy water can antagonise effects of general anæsthetics. It is suggested that microtubular proteins are also susceptible to reversible depolymerisation by anæsthetics, and that this is achieved by binding to non-polar sites on globular proteins. How this depolymerisation results in narcosis is not yet clear, but one possibility (suggested by the abundance of microtubules in neurons) is that microtubules participate in nerve-impulse transmission.

Structure and function of haemoglobin: II. Some relations between polypeptide chain configuration and amino acid sequence

X-Ray data suggest that the globin chain has the same configuration in the myoglobins and haemoglobins of all vertebrates. Sequence data, on the other hand, show that only 9 out of more than 140 sites are occupied by the same amino acid residue in all the globin chains analysed so far. The different globins do not have any marked pattern of ionized or of polar residues in common. The most prominent common feature is the almost total exclusion of polar residues from the interior of the globin chains; this expresses itself in a pattern of 30 sites where only non-polar residues occur. Along α -helical segments these invariant non-polar sites tend to repeat on the average at regular intervals of about 3·6 residues, making the interior face of the helix non-polar. At most sites at the surface or in surface crevices, on the other hand, replacements of many different kinds seem to occur without affecting the tertiary structure: these include replacement of non-polar by polar residues and vice versa . Prolines are confined to the ends of helices or to non-helical regions; otherwise their incidence in the globins of different species is largely random. If all the proline sites observed are plotted along the sequence, they are seen to place limits on the possible lengths of helical regions. In many instances, serine, threonine, aspartic acid or asparagine occupies the first site at the amino end of the helix, followed by a proline at the second site. In a protein of unknown structure, a regular periodicity of invariant non-polar sites might serve to recognize helical regions, and the incidence of prolines to define their lengths. A common pattern of non-polar sites might also help to identify structurally similar proteins with different enzymic function.

The interaction of a naphthalene dye with apomyoglobin and apohemoglobin: A fluorescent probe of non-polar binding sites

1-Anilino-8-naphthalene sulfonate binds stoichiometrically to a specific site on apomyoglobin and apohemoglobin. One mole of ANS† is bound per mole of apoprotein with a dissociation constant of the order of 10 −5 M . Myoglobin and hemoglobin do not bind ANS. The addition of hemin displaces it from its complex with apomyoglobin, suggesting that ANS and heme bind at the same site. Its fluorescence changes markedly when it is bound to the apoprotein. The quantum yield is two-hundredfold higher than in water, while the emission peak is shifted from green to blue by 60 m μ . These changes are attributed to the essentially non-polar environment of the bound ANS. A similar dependence of quantum yield and emission maximum on the polarity of the environment is observed for solutions of ANS in various organic solvents. Fluorescence polarization and optical rotatory dispersion results indicate that the compactness and high helix content of myoglobin are retained in large part in the apomyoglobin-ANS complex. Highly efficient energy transfer is observed from the aromatic amino acids of the protein to the bound ANS. The use of anilinonaphthalene sulfonates as fluorescent probes of non-polar regions in proteins is discussed.