Mechanistic differences in the energy-linked fluorescence decreases of 9-aminoacridine dyes associated with bovine heart submitochondrial membranes

(1) The pH dependence of the fluorescence intensities of 9-aminoacridines associated with energized submitochondrial membranes suggests that a mechanism(s) other than protonation of the dye molecules, as is the case with quinacrine, is responsible for the energy-linked fluorescence decreases of 9-aminoacridine and 9-amino-3-chloro-7-methoxyacridine (9-ACMA). (2) That the fluorescence polarization of quinacrine associated with submitochondrial membranes more than doubles upon energization of the membranes is attributed to: (i) the bulky side chain at the 9-position of the acridine moiety which hinders the molecular rotation of quinacrine and (ii) electrostatic forces resulting from the protonation of quinacrine . H+ which induce tight binding between the dye molecules and the membranes. (3) The protonation of quinacrine associated with energized membranes, from the monoprotonated to the diprotonated species, takes place in the membrane phase, as evidence by the observation of a 'break' in both the Arrhenius plot of the respiratory rate and the plot of fluorescence polarization as a function of temperature. (4) That the measured fluorescence polarization of both 9-aminoacridine and 9-ACMA associated with both energized and nonenergized membranes is nearly zero suggests that the emitting species of these dye molecules are those in the 'free' form and that the membrane-bound molecules have formed nonfluorescent complexes; consequently no polarization can be measured.     

Photoaffinity labelling of submitochondrial membranes with the 3-azido analogue of 9-amino-3-chloro-7-methoxyacridine

9-Amino-3-azido-7-methoxyacridine has been synthesized and shown to be a suitable photoaffinity probe for the site(s) of interaction of 9-amino-3-chloro-7-methoxyacridine with submitochondrial membranes. Both the excitation and emission spectra of the azido analogue covalently bound to membranes in the energized state display distinctive differences from the spectra of labelled, non-energized membranes (i.e., in the absence of oxidizable substrate, or its presence when uncoupler (FCCP) is also present during photolysis). Enzymatic analyses indicate that the probe interacts with the ATPase and the respiratory chain enzymes; energization appears to afford some protection against inactivation. Electrophoresis of the labelled membranes and isolation of their lipid and protein components indicate that the spectral differences are attributable to differing interactions with the lipid components of energized, relative to non-energized, membranes. Similar results have been obtained with the 3-azido analogue of quinacrine (Mueller, D.M., Hudson, R.A. and Lee, C.P. (1982) Biochemistry 21, 1445-1453), which differs significantly, however, in the extent of its interactions with the enzymes of the respiratory chain and the ATPase. These results indicate that the energy-linked fluorescence responses of 9-aminoacridines with submitochondrial membranes arise from direct interactions with membrane components and may involve redistribution of the probe molecules and/or alteration of their microenvironments upon energization.     

Fluorescence of quinacrine mustard conjugated to proteins.

Proteins are readily labeled by quinacrine mustard to yield conjugates whose spectral properties are well-suited for fluorescence studies. Data on these conjugates and on the parent compound, quinacrine, are presented including lifetimes, quantum yields, and corrected excitation and emission spectra. Polarization studies using the Perrin-Weber equation show that rotational relaxation times can be obtained with quinacrine mustard conjugates. Such conjugates had lifetimes ranging from 4 to 13 ns and quantum yields from about 0.1 to 0.3. Quinacrine mustard is a useful reporter group, as shown by the changes in fluorescence parameters of conjugates undergoing conformational changes induced by denaturants. An excited state $\text{p}\text{K}{}_{\mathrm{a}}{}^1$ of 4.9 was identified for quinacrine, but the protonation was suppressed in the mustard conjugate of serum albumin until the N-F transition took place. The properties of the mustard conjugates are discussed in terms of potential uses and compared with properties of other types of fluorescent conjugates.     

Fluorescence and phosphorescence of adrenolutin

Adrenolutin 3,5,6-trihydroxy-1-methylindole is an intermediate in the metabolism of adrenaline and in the formation of adrenochromo-melanins. Excitation and emission spectra, quantum yield of the adrenolutin fluorescence in water, D2O, ethanol, methanol, acetone and aqueous phosphate buffer at different pH at 293K temperature are reported. Dependence of the quantum yield of adrenolutin on its concentration are measured. Lifetimes of 0.1 mM adrenolutin in water and ethanol are 32.0 +/- 0.2 ns and 9.2 +/- 0.2 ns respectively. Also fluorescence and phosphorescence spectra of adrenolutin in methanol at 110K are obtained. Degrees of polarization and angles between the dipoles for the three main bands absorption of adrenolutin from measurements at 103K are calculated. Adrenolutin may be classified as one of the most strongly fluorescing metabolites. Broad excitation spectrum and high quantum yields make this compound a potential effective acceptor of excitation energy.     

Gradation of the magnitude of the electrochemical proton gradient in Mycoplasma cells

The results presented show that in Mycoplasma mycoides var. Capri, regulation of glucose uptake by its non-metabolizable analogue methyl alpha-D-glucoside, can be used to control intracellular ATP content. This in turn leads to a control of the rate of proton extrusion catalysed by the Mg2+-dependent ATPase (phi (cHxN)2C H+) and the respective amplitudes of the components of delta mu H+. When Mycoplasma cells are incubated with 10 mM methyl alpha-D-glucoside, the amplitude of phi (cHxN)2C H+, of the electrical potential delta psi and of the chemical gradient delta pH become continuous functions of external glucose concentration within the limits of the non-energized and fully energized states. Analysis of the relationships between graduated amplitudes of delta psi, delta pH and phi (cHxN) 2C H+ show that the primary form of energy stored by a delta mu H+ generator is the electrical potential.     

Luminescence of bacteriorhodopsin from Halobacterium halobium and its connection with the photochemical conversions of the chromophore

Red luminescence of purple membranes from Halobacterium halobium cells in suspension, dry film or freeze-dried preparations was studied and its emission, excitation and polarization spectra are reported. The emission spectra have three bands at 665–670, 720–730 and at 780–790 nm. The position (maximum at 580 nm) and shape of the excitation spectra are close to those of the absorption spectra. The spectra depend on experimental conditions, in particular on pH of the medium. Acidification increases the long wavelength part of the emission spectra and shifts the main excitation maximum 50–60 nm to the longer wavelength side. Low-temperature light-induced changes of the absorption, emission and excitation spectra are presented. Several absorbing and emitting species of bacteriorhodopsin are responsible for the observed spectral changes. The bacteriorhodopsin photoconversion rate constant was estimated to be about 1 · 10¹¹ s^?1 at ? 196°C from the quantum yields of the luminescence (1 · 10^?3) and photoreaction (1 · 10^?1). The temperature dependence of the luminescence quantum yield points to the existence of two or three quenching processes with different activation energies. High degree of luminescence polarization (about 45–47%) throughout the absorption and fluorescence spectra and its temperature independence show that there is no energy transfer between bacteriorhodopsin molecules and no chromophore rotation during the excitation lifetime. In carotenoid-containing membranes, energy migration from the bulk of carotenoids to bacteriorhodopsin was not found either. Bacteriorhodopsin phosphorescence was not observed in the 500–1100 nm region and the emission is believed to be fluorescence by nature.     

Compositional heterogeneity reflects partial dehydration in three-dimensional crystals of bacteriorhodopsin

Absorption, fluorescence and excitation spectra of three-dimensional bacteriorhodopsin crystals harvested from a lipidic cubic phase are presented. The combination of the spectroscopic experiments performed at room temperature, controlled pH and full external hydration reveals the presence of three distinct protein species. Besides the well-known form observed in purple membrane, we find two other species with a relative contribution of up to 30%. As the spectra are similar to those of dehydrated or deionized membranes containing bacteriorhodopsin, we suggest that amino acid residues, located in the vicinity of the retinal chromophore, have changed their protonation state. We propose partial dehydration during crystallization and/or room temperature conditions as the main source of this heterogeneity. This assignment is supported by an experiment showing interconversion of the species upon intentional dehydration and by crystallographic data, which have indicated an in-plane unit cell in 3D crystals comparable to that of dehydrated bacteriorhodopsin membranes. Full hydration of the proteins after the water-withdrawing crystallization process is hampered. We suggest that this hindered water diffusion originates mainly from a closure of hydrophobic crystal surfaces by lipid bilayers. The present spectroscopic work complements the crystallographic data, due to its ability to determine quantitatively compositional heterogeneity resulting from proteins in different protonation states.     

Characteristics of the tertiary structure of histone H1 from the calf thymus

By optical methods it has been previously shown that the globular "head" of histone H1 forms a hydrophobic cavity containing Tyr72. The latter is screened from the polar water surrounding and its intramolecular mobility is drastically hindered. As a consequence of the alteration in the micromilieu are a long wave shift (lambda max = 279,5 nm) and a more pronounced longwave absorption spectra, higher anisotropy (A = 0,11), augmented quantum yield of fluorescence (approximately 0,2) and a decrease of the Stern-Volmer constant for Hl at fluorescence quenching by acrylamide. It was found that changes in fluorescence intensity of histones are connected with alterations in the quantum yield of fluorescence at lambda exc = = 265 nm, but not at lambda exc = 280 nm. The changes in fluorescence intensity at light excitation 280 nm (F280) and 265 nm (F265) are in good accordance with shift delta E286 in differential absorption spectra. Introduction of parameter Cf = F280/F265 allows to study shifts of excitation spectra instead of shifts in absorption spectra of histones. This method has certain advantages, since it permits investigations with lower protein concentrations and in turbid solutions. The data obtained allow to draw out Tyr72 of histone Hl into a special class of fluorescent-tyrosyls, that differ in properties from those of other tryptophandevoided proteins: RNAse, insulin and core-histones H2A, H2B, H3 and H4.     

Synthesis and spectral studies of 2-[(N-ethyl carbazole)-3-sulfonyl ethylenediamine]-1-N,N-2-(2-methypyridy) as a fluorescence probe for Zn²⁺

A novel Zn(2+) fluorescence probe, 2-[(N-ethyl carbazole)-3-sulfonyl ethylenediamine]-1-N,N-bis(2-methypyrbidy), was designed and synthesized via simple steps, and its structure was confirmed by IR and (1)H NMR. The probe gives significant fluorescence enhancement immediately following Zn(2+) addition at neutral pH and exhibits improved selectivity for Zn(2+) compared to the other metal ions in aqueous solution. The spectra and fluorescence quantum yield of the synthesized compound were carefully investigated by UV-vis absorption and fluorescence spectra in various solvents.     

Excited-state dynamics of bacteriorhodopsin.

Near infrared emission of bacteriorhodopsin at neutral pH and at room temperature was characterized by a large Stokes shift. This characteristic was lost in an acidic pH (approximately pH 2) where a remarkable enchancement (more than 10 times) in the fluorescence quantum yield accompanied the red shift in the main absorption band. It is suggested from fluorescence polarization measurements that the emission occurs from the first allowed excited state of the retinylidene chromophore, irrespective of pH. We suggest that the large Stokes shift observed at neutral pH is a result of a charge displacement (e.g., proton translocation) that occurs immediately after excitation, and is prevented by protonation (in the ground state) of an amino-acid residue in the protein.     

Quinacrine inhibits the calcium-induced calcium release in heavy sarcoplasmic reticulum vesicles

Quinacrine is a fluorescence probe useful for studying the effect of local anesthetics. The interaction of quinacrine and sarcoplasmic reticulum membranes measured by fluorescence spectroscopy indicates the presence of a saturable binding site. Typical local anesthetics are able to displace quinacrine bound to heavy sarcoplasmic reticulum membranes. The effectiveness of that displacement decreases in the order dibucaine greater than tetracaine greater than benzocaine greater than lidocaine greater than procaine greater than procainamide, indicating that the size and hydrophobicity of quinacrine are major determinants in the binding process. The use of radioactive tracer and a rapid filtration technique reveals that quinacrine interacts, at lower concentrations, with sarcoplasmic reticulum membranes by blocking the Ca2+-induced Ca2+ release. Higher quinacrine concentrations also affect the Ca2+-pump activity.     

Proton movements coupled to sugar transport via the galactose transport system in Salmonella typhimurium.

We have studied proton movements associated with substrate transport via the galactose transport system in Salmonella typhimurium. The addition of galactose to lightly buffered suspensions of anaerobic, non-metabolizing cells of Salmonella typhimurium, specifically induced for the galactose transport system, causes an increase in extracellularpH as galactose and protons enter the cell together. Other substrates for this transport system, D-fucose, 2-deoxygalactose, glucose and 2-deoxyglucose similarly cause an influx of protons when transported. In contrast, transport via the other major transport system for galactose, the methylgalactoside transport system, is not coupled to H+ influx. Comparison of kinetic data obtained from pH measurements with data obtained from measurement of active transport of galactose via the galactose transport system suggests that the apparent Km of the galactose transport system for this sugar differs under energized and non-energized conditions. At pH 7.2 the permeant anion SCN- increases both the rate and extent of galactose-induced proton influx; at pH 6 the rate, but not the extent is increased by SCN-.     

Resonance Raman evidence for low-spin Fe2+ heme a3 in energized cytochrome c oxidase: implications for the inhibition of O2 reduction

Resonance Raman (RR) spectra are reported for reduced submitochondrial particles (SMP) with excitation at 441.6 nm, where Raman bands of the cytochrome c oxidase heme a groups are selectively enhanced. Addition of ATP to energize the membranes induces the formation of a new band at 1644 cm-1 and partial loss of intensity in a band at 1567 cm-1. These changes are modeled by adding cyanide to reduced cytochrome c oxidase and are attributed to partial conversion of cytochrome (cyt) a3 from a high-spin to a low-spin state. This conversion is abolished by addition of excess oligomycin, an ATPase inhibitor, or FCCP, an uncoupler of proton translocation, and is reversed when the ATP is consumed. The observed spin-state conversion is attributed to the binding of an endogenous ligand to the cyt a3 Fe atom. This ligation is suggested to be induced by a local increase in pH and/or by a global conformation change associated with the generation of a transmembrane potential. Since O2 binding requires a vacant coordination site at cyt a3, the ligation of this site must retard O2 reduction and could thus provide a simple mechanism for energy-linked regulation of respiration. No changes in the RR spectrum were observed upon adding Ca2+ or H+ to reduced cytochrome c oxidase. The cyt a3 spin-state change associated with membrane energization is unrelated to the cyt a absorption red shift induced by adding Ca2+ or H+ to cytochrome c oxidase.     

Interaction of aurovertin with submitochondrial particles,deficient in ATPase inhibitor

1. Binding of aurovertin to submitochondrial particles deficient in ATPase inhibitor is accompanied by an enhancement of the fluorescence by at least 100-fold.2. This change in fluorescence proceeds in three phases. The slowest change may be due to a conformational change in F₁, induced by the antibiotic bound during the rapid phases, giving rise to an increase in the quantum yield of the bound fluorochrome.3. Phosphate and ATP quench the fluorescence of the particle-aurovertin complex and ADP enhances it; the rate and extent of these changes are dependent on the availability of free Mg²⁺.4. There is at least one binding site on the submitochondrial particles, where ATP, ADP and phosphate can bind reversibly and for which these ligands compete. These interactions are dependent on the availability of free Mg²⁺ and are partly sensitive to oligomycin.5. Binding studies reveal two binding sites for aurovertin on inhibitor-free particles, one with high affinity and one with a lower affinity. Ligands such as phosphate and ATP decrease both the quantum yield and the affinity of the particles for aurovertin. They also increase the total concentration of binding sites, and affect the relative contribution of weak and strong binding sites.6. A model is presented in which changes of the aurovertin fluorescence reflect conformational changes of the ATPase induced by its ligands.     

Modification of the biosynthesis and composition of polyglycerophosphatides in outer and inner mitochondrial membranes by cytidine liponucleotides

The biosynthesis of [3H]polyglycerophosphatides ([3H]phosphatidylglycerophosphate and [3H]phosphatidylglycerol) in mitochondrial and submitochondrial (outer and inner) membranes isolated from guinea pig liver was examined. Experimental results have established that the amount of biosynthesized [3H]polyglycerophosphatides and the relative amounts of biosynthesized [3H]phosphatidylglycerol and [3H]phosphatidylglycerolphosphate can be influenced by varying the composition of fatty acids in CDP-diglycerides and by altering the incubation time of the mixture containing CDP-diglycerides (obligatory precursor), sn-[2-3H]glycerol-3-phosphate and mitochondria or submitochondrial membranes. The changes thus obtained in respect to the amount and composition of biosynthesized [3H]polyglycerophosphatides are different in mitochondria and submitochondrial membranes. The highest amount of biosynthesized [3H]polyglycerophosphatides was obtained with CDP-didecanoin and inner mitochondrial membranes. The greatest accumulation of [3H]phosphatidylglycerol with CDP-didecanoin was obtained in mitochondria and outer mitochondrial membranes, while in inner mitochondrial membranes the amounts of [3H]phosphatidylglycerol and [3H]phosphatidylglycerolphosphate accumulated were approximately the same. In general, prolongation of the incubation time decreased the relative amounts of [3H]phosphatidylglycerolphosphate and increased the amount of accumulated [3H]phosphatidylglycerol, but the absolute amounts of these [3H]polyglycerophosphatides were more dependent on fatty acid composition of CDP-diglycerides tested. The following cytidine liponucleotides were tested: CDP-didecanoin, CDP-dipalmitin, CDP-diolein, and CDP-diglycerides containing saturated and unsaturated fatty acids similar to those in egg yolk lecithin. The formation of [3H]cardiolipin from [3H]phosphatidylglycerol in the presence of CDP-didecanoin and Mn2+ was found in both the outer and inner mitochondrial membranes.     

Study of pH-dependence of kinetic parameters of pyruvate kinase from bovine adrenal cortex

The effect of pH on the main kinetic parameters of pyruvate kinase function was studied. The maximal rate of the reaction as well as the values of Km for ADP and Ki for phenylalanine depend on pH and show a well-defined extremum at pH 6.8-7.0. Spectrofluorimetric titration of pyruvate kinase results in pH dependencies of changes in the fluorescence spectra parameters (e.g., quantum yield, half-width and position of the maximum). This enabled to determine the pH regions corresponding to changes in the state of tryptophan residues. Data from the enzyme inhibition by phenylalanine suggest that acidification of the medium leads to the decrease of the catalytic activity due to the protonation of the ionogenic group of the enzyme. Within the pH range of 7.0-8.0, the decrease of the pyruvate kinase activity is due to structural shifts in the enzyme molecule, as a result of which the steric complementariness of the enzyme active center with respect to the substrate (Mg.ADP) is impaired.     

The fluorescence quantum yield of vitamin A2

The fluorescence quantum yield of all-trans 3,4-didehydroretinol (vitamin A2) was measured in hexane at room temperature, using quinine sulfate as a standard. Unlike all-trans retinol (vitamin A1) which possessed a relative quantum yield of 0.0298, 3,4-didehydroretinol was 37 times lower in fluorescence (i.e. 0.0008). In addition, a significant bathochromic shift (both excitation and emission maxima) and a general broadening of the fluorescence spectra were noted for 3,4-didehydroretinol. This information is important not only for the understanding of the basic structure of vitamin A but also the photochemistry of vision.     

Fluorescence of myosin and its subunits in concentrated salt solutions]

The parameters of fluorescence spectra of myosin and its subunits in Tris-HCl-buffer (pH 7.2) were studied. Analysis of the experimental results of myosin fluorescence quenching with I-ions and the quantum yield of the fluorescence at the excitation wavelength 296 nm shows that the greater part of the tryptophan residues (21 out of 28) is located in the hydrophylic environment. Concentrated solutions of NaCl and KCl do not affect myosin fluorescence, while LiCl, which changes the quaternary structure of the protein, brings about a change in the parameters of the myosin fluorescence spectra. This may be linked with structural changes accompanying the dissociation of the ligh subunits of myosin in the presence of LiCl.     

Mechanism of acridine uptake by submitochondrial particles.

Acridines were compared regarding their ability to be taken up by submitochondrial particles under energized conditions. pH dependence of uptake was explored, and it was found that acridines fell into three classes independently of their pK_a value: acridines which are not taken up, acridines taken up at all pH values, and acridines taken up only at alkaline pH. Partition measurements between H₂O and chloroform phase showed a similar pattern, and affinity for the organic phase seemed to parallel uptake. Acridines which are taken up by submitochondrial particles at acidic pH under energized conditions despite a high pK_a value could also be extracted into chloroform at acidic pH, thus implying that the dye's uncharged form has a high affinity for the organic phase. By supplementing the aqueous medium with lipophilic anions, the dye may also be extracted in its charged form. The data support a mechanism for acridine uptake in which diffusion of the uncharged form across the membranes is an obligatory step. Some previously reported inhibitory anion effects on uptake may be explained by ion pair formation, which allows release of the accumulated charged form.