SALT AND pH-DEPENDENT CHANGES OF THE PURPLE MEMBRANE ABSORPTION SPECTRUM

Abstract —Purple membrane suspensions change their color to blue and the absorption maximum shifts to 608 nm when the membrane is deionized on a cation exchange column or when it is washed first with < 2N NaCl followed by deionized water. The deionized chromophore is essentially identical with the chromophore produced by lowering the pH of the native membrane to < 4.0 (p K < 3.0). However, the deionized membrane does not aggregate and can be obtained in the pure state. The original purple color of the membrane is restored by addition of around 1 m M Na⁺, K⁺ or 10 μ M Mg²⁺, Ca²⁺, Sr²⁺, Mn²⁺, Pb²⁺ or La²⁺ when the protein concentration is 5μ M . The required salt concentrations decrease with decreasing pH. Direct measurement of bound Ca²⁺ by atomic absorption spectroscopy yields a ratio of Ca²⁺ to protein of <2 and a binding constant of 1.4 × 10⁶. Titration of the spectral change with salts at different pH values shows a linear relation between the pH and the logarithm of the salt concentration, with a 1:1 ratio for Na⁺ and 1:2 ratio for Ca²⁺. These relations are well predicted by Gouy-Chapman theory; however, the accompanying release of protons, changes of the CD spectrum, the complex kinetics of the spectral change during reconstitution with salt and preliminary X-ray diffraction results all suggest that conformational changes may be occurring in the protein.     

DETERMINATION OF THE pk VALUES OF THE LUMIFLAVIN TRIPLET STATE BY FLASH PHOTOLYSIS

Abstract— The triplet-triplet absorption spectra in aqueous solution of the acid (³LfH₂⁺), the neutral (³LfH) and the basic (³Lf^-) forms of lumifiavin (6,7,9-trimethylisoalloxazine) were measured by flash photolysis. The p K _a values of the corresponding protolytic equilibria of the lumifiavin triplet were found to be 4.4₅±0.1 and 9.8±0.15.     

REEVALUATION OF THE SPECTRAL AND KINETIC PROPERTIES OF HO2 AND O2- FREE RADICALS

Abstract— The spectra and molar absorbances of the HO₂ and O₂^- free radicals have been redetermined in aqueous formate solutions by pulse and stopped-flow radiolysis as well as by ⁶⁰Co gamma-ray studies. The extinction coefficients at the corresponding maxima and 23°C are ²²⁵= 1400 ± 80 M ^-1 cm^-1 and ²²⁵= 2350 ± 120 M ^-1 cm^-1 respectively. Reevaluation of earlier published rate data in terms of the new extinction coefficients yielded the following rate constants for the spontaneous decay of HO₂ and O₂^-: K _Ho2+HO2= (8.60 ± 0.62) × 10⁵ M ^-1 s^-1; K _Ho2+O2-= (1.02 ± 0.49) × 10⁸ M ^-1 s^-1; K _Ho2+O2- < 0.35 M ^-1 s^-1. For the equilibrium HO₂→ O₂^-+ H⁺ the dissociation constant is K _Ho2= (2.05 ± 0.39) × 10^-5 M or p K _HO2= 4.69 ± 0.08. G (O₂^-) has been evaluated as a function of formate concentration.     

ELECTRON TRANSFER FROM THE EXCITED STATE OF TYROSINE TO COMPOUNDS CONTAINING DISULFIDE LINKAGES

Abstract— The flash photolysis of aqueous solutions of tyrosine has been studied in the presence of various concentrations of the cyclic disulfide sodium lipoate (thioctic acid, Na⁺ salt). In addition to the formation of phenoxyl radicals and hydrated electrons (and possibly H atoms) from the photoionization of tyrosine, the characteristic spectrum of the radical anion RSSR^- of lipoate was also observed in neutral as well as in alkaline solutions. From the dependence of these yields upon the concentration of lipoate, it was found that a long–lived triplet excited state of tyrosine, rather than the singlet excited state, is involved in these reactions. The negative radical ions RSSR^- are formed by two distinct pathways: (a) Na⁺–lipoate reacts with the solvated electrons which are ejected from the tyrosine triplets ³Tyr → RO.+ e ^-_aq+ H⁺ followed by e ^-_aq+ RSSR → RSSR^-, and (b) by direct interaction of lipoate with triplet excited tyrosine, resulting in the transfer of a negative charge from tyrosine to the disulfide linkage. At high lipoate concentrations, the singlet excited state of lipoate is quenched, k ₄= 1.6 × 10¹⁰ M ^-1 sec^-1, but this reaction does not lead to the formation of RSSR^- radical ions.     

BLUE LIGHT INDUCED, REVERSIBLE IN ACTIVATION OF THE TONOPLAST-TYPE H+-ATPase FROM CORN COLEOPTILES IN THE PRESENCE OF FLAVINS

Abstract— Irradiation (λ_max 447 nm; 58.5 W m^-2) of a microsomal membrane fraction of corn coleoptiles for 5 min in the presence of the in vivo concentration of riboflavin inactivates the tonoplast-type H⁺-ATPase. This inhibition is O₂-dependent, is enhanced in D₂O and suppressed by NaN₃, indicating participation of singlet molecular oxygen in the inactivating mechanism. Besides singlet oxygen, the superoxide anion (O₂^-) is generated during irradiation, which obviously has no effect on the H⁺-pumping activity. However, in the presence of superoxide dismutase (SOD), O₂^- is transformed into H₂O₂ which causes an additional strong inhibition of H⁺. ATPase activity. This inhibition can be increased by ethylenediaminetetraacetic acid (EDTA), which is known to be an electron donor of the excited flavin molecule. In contrast, catalase prevents the H₂O₂-mediated photoinactivation of the H⁺ -ATPase. The light dependent inactivation of H⁺-transport does not occur if reduced glutathion (GSH) is added prior to or after irradiation. These results indicate that the blue light mediated inhibition of the H⁺-ATPase is mediated by singlet oxygen and H₂O₂ which oxidize essential SH-groups of the enzyme into disulfides. Reduction of the formed disulfides by GSH restores the activity of the enzyme.     

温度和离子对紫膜LB膜光循环的影响

用闪光动力学光谱仪测量了水平拉制的紫膜LB膜中菌紫质中间体M₄₁₂的衰减过程,观察了温度和离子对M₄₁₂衰减过程的影响。实验结果表明:在一定的温度范围内(10℃-60℃),随着温度的升高,M₄₁₂的衰减速率加快。对M₄₁₂s的衰减的抑制作用,La³⁺在低浓度时就很明显,而K⁺则在较高浓度时才表现出来,Ca²⁺的影响不明显;La³⁺对M₄₁₂f的衰减无明显影响,K⁺和Ca²⁺则稍微加快了其速率,pH的变化(H⁺浓度)明显影响到M₄₁₂的衰减速率,尤其在高pH情况,M₄₁₂s的衰减比正常pH值时要慢一个数量级。     

A FLASH PHOTOLYSIS STUDY OF 5-METHOXYINDOLE

Abstract— The microsecond flash photolysis of 5-methoxyindole in aqueous solutions has been studied at γ_exc≥ 290 nm. Transients identified in this time realm in neutral solutions are: e_aq^-, the 5-methoxyindole radical cation (γ_max≅ 440 nm), the neutral transient with γ_max≅ 530 nm) and an unidentified oxygen sensitive transient with γ_max≅ 435 nm. Radical cations and e^-_aq are shown to be produced in equal amounts consistent with a photoionization process as the only source of both transients. H⁺ quenching of fluorescence and radical cation production gives equivalent Stern-Volmer constants indicating that photoionization occurs from the fluorescent state. The unidentified oxygen sensitive transient exhibits a pK _a of2–2.5 and is quenched at lower pH values indicating that it also has a fluorescent state precursor.     

RATE CONSTANTS FOR INTERACTION OF 1O21Δg) WITH AZIDE ION IN WATER

Abstract— The rate constant for quenching of ¹O₂ by azide ion in water was determined to be (5.0 ± 0.4) × 10⁸ M ⁻¹ s⁻¹ using a variety of sensitizers (including humic acids) and ¹O₂ acceptors. The apparent second-order rate constant decreases with pH below pH 5.5 in accordance with the protonation of azide ion to form hydrazoic acid (p K _a= 4.6). Quenching by hydrazoic acid is at least 2 orders of magnitude slower than by azide ion. Greater than 99% of all interactions between ¹O₂ and azide ion involve physical quenching rather than chemical reaction. Humic acid triplets are not significantly quenched by azide ion at concentrations less than 2 m M , allowing azide ion quenching to be used as a diagnostic test for the intermediacy of ¹O₂ in photosensitized oxidations in natural surface waters.     

STOICHIOMETRY and KINETICS OF PROTON RELEASE UPON PHOTOSYNTHETIC WATER OXIDATION

Abstract— We studied the magnitude and the rise kinetics of proton release into the interior of thylakoids by flash spectrophotometty with neutral red as pH indicator. Excitation of dark-adapted thylakoids by a series of between 4 and 11 flashes produced a complex pattern of proton release into the thylakoid lumen. Proton release upon each flash was time resolved.
A slow component of proton release oscillated weakly in magnitude with period of two as function of flash number. It exhibited a half-rise time of approximately 20 ms from the very first flash on, and it was abolished by inhibitors of plastohydroquinone oxidation. This component was attributed to the oxidation of plastohydroquinone by PS I via the Cytb₆/f complex.
Additionally, rapid and multiphasic proton release was observed with half-rise times of less than 2 ms which exhibited a pronounced and damped oscillation with period of four as function of flash number. This rapid proton release was attributed to water oxidation. A detailed kinetic analysis suggested that proton release occurred with the following stoichiometry and with the following half-rise times during the transitions S₁ S_i+1 of water oxidation: 1 H⁺(250 μs, S₀→₁): 0 H⁺(S₁→ S₂):1 H⁺(200 μs, S₂→S₃):2 H⁺(1.2 ms, S₃→ S₄→ S₀) . Proton release and proton rebinding upon oxidation and reduction of the intermediate electron carrier Z, respectively, may have influenced the kinetics of the respective proton yields but not the stoichiometric pattern.     

A FLASH PHOTOLYSIS STUDY OF 1-METHYLINDOLE

Abstract— The conventional flash photolysis of 1-methylindole in aqueous media was studied at Λ_excitation≥290 nm. The transients observed 20 μs after excitation consisted mainly of the radical cation (R⁺). the hydrated electron (e^-_aq) and the triplet state (T). Electron counting experiments indicate that photoionization is the only source of R⁺ with e^-_aq/R⁺= 1.07±0.09 in neutral media. Quenching of the R⁺ yield with H⁺ indicates that the fluorescent state is the precursor to 80% of the photoionization events with the remainder probably arising from a prefluorescent state. The triplet decays with a lifetime of 29 μs in deaerated neutral media. This decay is unchanged by N₂O saturation, but T reacts with acrylamide with k ≥2.8 × 10⁹ M ^-1. In 2 M Br^-, R⁺ and T yields are increased by factors of 2–3. Consideration of fluorescence quenching and T enhancement by Br-permits an estimate of φ_Isc between 0.33 and 0.49. The increased R⁺yield at high Br-concentrations cannot be accounted for by induced photoionization or triplet state reactions.     

REACTIVITY OF ACRIDINE DYE TRIPLET STATES IN ELECTRON TRANSFER REACTIONS

Abstract—Rate constants, k _q , for the reaction of cationic and neutral acridine orange and 10-methylacridine orange triplet states (³AOH ⁺, ³AO, ³MAO⁺) with a series of electron donors have been measured. Two different protolytic forms of the semireduced dye radical are produced by acridine orange triplet quenching at various pH^M values in methanolic solution.
It is found that k ₄ decreases with increasing oxidation potential of the reducing agent for all triplet states in a manner which is expected for electron transfer reactions. The different reactivities of the cationic and neutral triplet forms can, therefore, be attributed to the difference in reduction potentials of these species. The difference in reduction potentials is related to the p K ^M values of triplet state, p K _T^M , and semireduced dye radical, p K ^M_S , by thermodynamic consideration. p K _T^M (³AOH⁺/³AO) is determined to be 11.2. From thisp K _S^M (AOH./AO;) is estimated to be 17–18. This is in striking contrast to the protolytic equilibrium of the semireduced dye radicals found to be pK^F= 4.1. We conclude that the last value represents the second protonation equilibrium (AOH⁺₂./AOH). This conclusion is confirmed by spectroscopic data.     

ÉTUDE EN SPECTROSCOPIE PAR ÉCLAIR DES COLORANTS THIAZINIQUES EN SOLUTION AQUEUSE

Abstract— p K values of ionisation equilibrium of thiazines dyes in their triplet state have been measured in aqueous solutions by spectroscopy. The triplet-triplet absorption bands, in the red part of the spectrum, are given for thionine, azurs and methylene blue. It is shown that, in the pH range 4–9, the equilibria investigated correspond to a second protonation of thiazines dyes that occurs in the triplet state:
³TH⁺+ H⁺→³TH₂²⁺
designating by ³TH⁺ the thiazine cation similar to the species stable in neutral aqueous solutions.     

BLEACHING OF PURPLE MEMBRANE WITH O-SUBSTITUTED HYDROXYLAMINES

Abstract The retinal Schiff base of bacteriorhodopsin, in the purple membrane from Halobacterium halobium , can be cleaved by hydroxylamine in the presence of light. We have further investigated this reaction with a series of O -substituted hydroxylamines, RONH₂, where R = -H (HA), -CH₃ (MHA), -SO₃− (HAS), benzyl- (BHA), p -nitrobenzyl- (NBHA), and pentafluorobenzyl- (FBHA). All except MHA caused light-induced bleaching of the purple membrane and the chromophore could be regenerated from apomembrane and all- trans retinal. Relative bleaching rate constants were obtained from V = QI _a k ₀ X /( k _r+ k ₀ X ), where V = bleaching rate, Q = quantum yield, I a = absorbed light intensity, X = hydroxylamine concentration, k ₀= rate constant for bleaching and k _r= rate constant for return of photoexcited bacteriorhodopsin to the initial state. This equation fits the time-, concentration- and intensity-dependences of the bleaching reactions in 0.02 M phosphate, pH 7.0. The rate constants k ₀ relative to HA were: MHA: 0; HAS: 0.3; HA: 1.0; BHA: 1.8; FBHA: 10.1; NBHA: 10.8. The relative rate constants do not correlate with the basicity of the derivatives. Instead, the results suggest that the retinal Schiff base is near a non-polar cavity into which an aromatic group can be inserted.     

THE ORIGIN OF THE RED-SHIFTED ABSORPTION MAXIMUM OF THE M412 INTERMEDIATE IN THE BACTERIORHODOPSIN PHOTOCYCLE

The factors that red shift the absorption maximum of the retinal Schiff base chromophore in the M₄₁₂ intermediate of bacteriorhodopsin photocycle relative to absorption in solution were investigated using a series of artificial pigments and studies of model compounds in solution. The artificial pigments derived from retinal analogs that perturb chromophore-protein interactions in the vicinity of the ring moiety indicate that a considerable part of the red shift may originate from interactions in the vicinity of the Schiff base linkage. Studies with model compounds revealed that hydrogen bonding to the Schiff base moiety can significantly red shift the absorption maximum. Furthermore, it was demonstrated that although s-trans ring-chain planarity prevails in the M₄₁₂ intermediate it does not contribute significantly (only ca 750 cm⁻¹) to the opsin shift observed in M₄₁₂. It is suggested that in M₄₁₂, the Schiff base linkage is hydrogen bonded to bound water and/or protein residues inducing a considerable red shift in the absorption maximum of the retinal chromophore.     

STUDY OF LUMINESCENT FORMS OF THE URANYL ION

Luminescence spectra and luminescence decay kinetics of uranyl sulphate water and uranyl nitrate acetone solutions of different concentrations have been studied. Similar experiments have been done with uranyl sulphate powder under vacuum. It has been experimentally shown that the hydrolysis of uranyl sulphate in water takes place, and under low salt concentrations (0.1-4.0 times 10^-4 M) a luminescence of a basic form of the photoexcited ion with a tentative structure of UO₂OH⁺* has been observed. The luminescence of the acidic form UO⁺* has been observed under higher salt concentrations (1–4 times 10^-2 M) in water and under any salt concentration in acetone. The acidic form has the characteristic emission spectrum possessing vibrational structure. The luminescence concentrational quenching of both photoexcited uranyl forms and exciplex emission have not been observed. The effect of a number of organic quenchers and molecular oxygen on uranyl luminescence has been studied. There is no luminescence quenching by O₂ up to 2 times 10⁶ Pa (20 atm) pressure. The low effectiveness of energy transfer from the photoexcited uranyl forms has been explained in terms of strong steric screening of 5f-uranium (VI) orbital by oxygen atoms and by external filled up uranium electronic shells.     

ACIDITY DEPENDENCE OF THE ABSORPTION AND FLUORESCENCE SPECTRA OF ISOFLAVONE AND 7-HYDROXYISOFLAVONE

-The pH and H₀ dependence of the absorption and fluorescence spectra of isoflavone and 7-hydroxyisoflavone are reported. Isoflavone is fluorescent in acidic solution only, whereas 7-hydroxyisoflavone is fluorescent in all acidity ranges under investigation. Ground and first excited singlet state p K _a's have been determined spectrophotometrically and fluorimetrically, respectively. Excited state protolytic equilibration processes via a second order reaction (proton gain) are found to be too slow to compete efficiently with fluorescence. This is deduced from the close agreement between the p K _a's of the conjugate acids obtained by absorption and fluorescence titrations. On the other hand, photodissociation of 7-hydroxyisoflavone proceeds faster than its fluorescence decays. The experimental p K _a(S₁) is in fair agreement with the calculated one. 7-Hydroxyisofiavone forms a phototautomer (or exciplex) in the pH 2 to H₀-1 acidity range, which is characterized by its long wavelength emission. Quantum efficiencies are given for isoflavone and 7-hydroxyisoflavone in aqueous solutions of various acidities. Deuteration effects thereon are discussed.     

THE QUANTUM EFFICIENCY FOR THE INTERPHOTOCONVERSION OF THE BLUE and PINK FORMS OF PURPLE MEMBRANE

When the cations bound to purple membrane are removed it turns blue, and when this blue membrane is irradiated its color changes to pink. Irradiation of pink membrane leads to the reformation of blue membrane. We have determined that the quantum efficiency for the formation of pink membrane from deionized blue membrane is 1.6 ± 0.6 ± 10 ⁴ at 0^oC, pH 5.0. We also found that the quantum efficiency for the back photoconversion, i.e. the formation of blue membrane from pink membrane, is 8.8 ± 1.6 ± 10^-3 at 0^oC, 55 times greater than that of the forward photoconversion reaction. The extinction coefficients of the pink membrane and blue membrane were determined to be 44 500 ± 670 cm^-1 M^-1 at 491 nm and 54 760 ± 830 cm^-1 M ^-1 at 603 nm, respectively, assuming light-adapted purple membrane is 63 000 cm^-1 M ^-1 at 568 nm. The quantum efficiency for forming pink membrane from blue membrane is much lower than that for forming the photointermediate of the blue membrane's photocycle. Their relationship is similar to that of light-adaptation and photocycle of the dark-adapted purple membrane.     

THE QUANTUM YIELD OF THE CHEMILUMINESCENCE OF DIMETHYLBIACRIDYLIUM NITRATE AND THE MECHANISM OF ITS ENZYMICALLY INDUCED CHEMILUMINESCENCE*

Abstract— Quantum yields for dimethylbiacridylium ion chemiluminescence based on the amount of methyl acridone formed by treatment with either H₂O₂ or with xanthine oxidasehypoxanthine in 0.01 M Na₂CO₃ at pH 10.4 and at 25°C were found to lie between 0.011 and 0.020 with an average of about 0.016.In mixed solvents containing pyridine and water or alcohol and water the emission spectrum of chemiluminescence of dimethylbiacridylium ions as well as those of diniethylbiacridene and its oxide were found to be identical with or very similar to the fluorescence of methyl acridone in the same solvent.A mechanism involving two successive two equivalent reductions of the diniethylbiacridylium ion to dimethylbiacridan followed by a radical attack and auto-oxidation leading to a compound which can undergo a reverse aldol type reaction to yield one (or two) molecule of methylacridone and one of a somewhat more reduced form is suggested.The kinetic equation relating maximum intensity of chemiluminescence to dimethylbiacridylium ion, hypoxanthine, xanthine oxidase, H⁺ and O₂ concentrations was derived from the scheme suggested and found to fit satisfactorily the data obtained from a series of experiments in which the quantum yield was also obtained.     

LIGHT-INDUCED PROTON DISSOCIATION AND ASSOCIATION IN BACTERIORHODOPSIN

Abstract— Light-induced proton release and uptake by acetylated and unmodified bacteriorhodopsin were measured. Bacteriorhodopsin, when illuminated, shows a net proton release at neutral and alkaline pH's, but in acidic pH, it shows an uptake of protons. In the presence of high concentrations of guanidine hydrochloride, light caused only proton release even in acidic pH and the maximum extent of the release was one proton per bacteriorhodopsin molecule around pH 8.
Acetylation of bacteriorhodopsin caused no alteration in the absorption spectrum of purple complex (bR₅₇₀) and M₄₁₂-intermediate, but decreased the decay rate of the M₄₁₂-intermediate. Light-induced release of protons was not observed even in neutral pH values, and only the proton uptake was noticed by acetylated purple membrane fragments. In high concentrations of guanidine hydrochloride, no proton uptake or release by illumination was observed. Vesicles were reconstituted from acetylated purple membrane. These vesicles had almost no ability for light-induced proton transport. The role of amino group(s) in light-induced proton release and transport through the purple membrane is discussed.     

SOLVENT EFFECTS ON THE PHOTOPHYSICAL PROPERTIES OF DIBUCAINE: A QUINOLINE ANALOGUE

Abstract— Emission and absorption spectra of neutral, monocation and dication dibucaine were recorded in hydrocarbon and hydroxylic solvents. The spectral analysis indicates that the lowest electronic states of dibucaine originate from the quinoline analogue in the molecule. The fluorescence and phosphorescence emission of neutral dibucaine in all solvents at 77 K are best assigned as resulting from ¹ n ,π* and ³π,π*, respectively, whereas those of both monocation and dication dibucaines are found to have ¹π,π* and ³π,π*. The monocation and neutral dibucaines in ethanol solutions were shown to give identical emission spectral properties, indicating that dibucaine-HCl in ethanol is H⁺ dissociative; this was confirmed by FT-IR studies. The possible explanations for the deprotonation of dibucaine-HCl in ethanol environments are discussed.