Modulation of Hydra attenuata rhythmic activity: phase response curve.

We investigated the effect of photic stimulation on the frequency of Hydra attenuata column contractions. We used positive or negative abrupt light transitions, single or repetitive light or darkness pulses, and alternation of light and darkness periods. The main results are: (a) The frequency of the contraction pulse trains (CPTs) varies transiently in response to an abrupt variation of the light intensity. (b) CPTs in progress can be inhibited by different types of photic stimuli. (c) The response time to a single photic stimulus varies during the inter-CPT interval and depends also on the polarity of the stimulus. (d) The CPTs are entrainable with repetitive light stimulation of various frequencies. (e) Long-lasting variations of the frequency of CPTs occur after the end of a repetitive light stimulation. We suggest that the mechanism responsible for the rhythym of column contractions is quite similar to that on which other biological rhythmic phenomena are based.     

Infrared spectroscopy of phytochrome and model pigments

Fourier-transform infrared difference spectra between the red-absorbing and far-red-absorbing forms of oat phytochrome have been measured in H2O and 2H2O. The difference spectra are compared with infrared spectra of model compounds, i.e. the (5Z,10Z,15Z)- and (5Z,10Z,15E)-isomers of 2,3,7,8,12,13,17,18-octaethyl-bilindion (Et8-bilindion), 2,3-dihydro-2,3,7,8,12,13,17,18-octaethyl-bilindion (H2Et8-bilindion), and protonated H2Et8-bilindion in various solvents. The spectra of the model compounds show that only for the protonated forms can clear differences between the two isomers be detected. Since considerable differences are present between the spectra of Et8-bilindion and H2Et8-bilindion, it is concluded that only the latter compound can serve as a model system of phytochrome. The 2H2O effect on the difference spectrum of phytochrome supports the view that the chromophore in red-absorbing phytochrome is protonated and suggests, in addition, that it is also protonated in far-red-absorbing phytochrome. The spectra show that protonated carboxyl groups are influenced. The small amplitudes in the difference spectra exclude major changes of protein secondary structure.     

pH-dependent reversible inhibition of violaxanthin de-epoxidase by pepstatin related to protonation-induced structural change of the enzyme

The redox enzyme violaxanthin de-epoxidase (VDE) was found to be sensitive to pepstatin, a specific inhibitor of aspartic protease. The inhibition was similar to that of aspartic protease in that it was reversible and accompanied by the protonation of the enzyme. Of the two peaks of VDE appearing on anion exchange chromatography, VDE-I predominated at pH 7.2. On lowering the pH of the chromatography, VDE-I decreased and VDE-II increased. Furthermore, re-chromatography of either peak yielded both peaks. These results suggest that VDE-I and VDE-II are interconvertible depending on pH, and thus, they represent the de-protonated and protonated forms of the enzyme, respectively. Presumably the protonation-induced structural change of the enzyme is responsible for the interaction with pepstatin, and also with substrate.     

Comparative structural analysis of 1-methyladenosine, 7-methylguanosine, ethenoadenosine and their protonated salts IV: 1H, 13C, and 15N NMR studies at natural isotope abundance.

The 1H, 13C, and 15N NMR spectra of neutral and protonated forms of the nucleosides 1-methyladenosine (m1A), 7-methylguanosine (m7G) and ethenoadenosine (EA), as a model compound, have been analyzed in order to assign the site of protonation in m1A and m7G. Protonation of these nucleosides occurs in the pyrimidine ring of m1A and EA and in the imidazole ring of m7G, with the charge being distributed rather than localized. Structural differences for both m1A and m7G were observed in solution and compared with those existing in the crystal state of monomers as well as in tRNA where these nucleosides occur quite often. The protonated nucleoside structures in solution compared favorably in sugar pucker and glycosidic bond conformations with x-ray crystallographic data. Methyl group carbon chemical shifts of the protonated mononucleosides corresponded to those of the methyls of the respective nucleosides in native tRNA structures. Therefore, the tRNA methyl group carbon chemical shifts are indicative of fully protonated nucleosides in the native, three dimensional structure of the nucleic acid.     

The pH-dependence of the binding of dihydrofolate and substrate analogues to dihydrofolate reductase from Escherichia coli

The interaction of dihydrofolate reductase (EC 1.5.1.3) from Escherichia coli with dihydrofolate and folate analogues has been studied by means of binding and spectroscopic experiments. The aim of the investigation was to determine the number and identity of the binary complexes that can form, as well as pKa values for groups on the ligand and enzyme that are involved with complex formation. The results obtained by ultraviolet difference spectroscopy indicate that, when bound to the enzyme, methotrexate and 2,4-diamino-6,7-dimethylpteridine exist in their protonated forms and exhibit pKa values for their N-1 nitrogens of above 10.0. These values are about five pH units higher than those for the compounds in free solution. The binding data suggest that both folate analogues interact with the enzyme to yield a protonated complex which may be formed by reaction of ionized enzyme with protonated ligand and/or protonated enzyme with unprotonated ligand. The protonated complex formed with 2,4-diamino-6,7-dimethylpteridine can undergo further protonation to form a protonated enzyme-protonated ligand complex, while that formed with methotrexate can ionize to give an unprotonated complex. A group on the enzyme with a pKa value of about 6.3 is involved with the interactions. However, the ionization state of this group has little effect on the binding of dihydrofolate to the enzyme. For the formation of an enzyme-dihydrofolate complex it is essential that the N-3/C-4 amide of the pteridine ring of the substrate be in its neutral form. It appears that dihydrofolate is not protonated in the binary complex.     

Optimized analytical method for cyclosporin A by high-performance liquid chromatography–electrospray ionization mass spectrometry

The Micromass Platform LCZ mass detector parameters were optimized for simultaneous recording of the protonated (CsA∼H⁺), sodium adduct (CsA∼Na⁺) and potassium adduct (CsA∼K⁺) of cyclosporin A eluted from a Symmetry Shield RP8 column. The optimized procedure allows a precise analysis of CsA in whole blood or serum without removal of salts prior to analysis. The ratio of the three forms of CsA varied depending on the assay condition and the types of specimens being analyzed. The summation of three ionic forms of CsA detected by LC–ESI-MS is a reliable and simple method to assess CsA concentration in the blood.     

The interaction between pyoverdin and its outer membrane receptor in Pseudomonas aeruginosa leads to different conformers: a time-resolved fluorescence study

In iron limitation conditions, Pseudomonas aeruginosa secretes a major fluorescent siderophore named pyoverdin (PaA). PaA has an extremely high affinity for Fe(3+) but also chelates other ions such as Al(3+) and Ga(3+) with a lower affinity. The transfer of PaA-Fe(3+) across the outer membrane of the bacteria is mediated by the receptor FpvA, a TonB-dependent outer membrane transport protein. FpvA binds the iron-free and iron-loaded forms of pyoverdin with similar affinities, but only PaA-Fe(3+) is taken up by the cell, suggesting that FpvA adopts different conformations depending on its loading status. We used time-resolved fluorescence spectroscopy to characterize the different forms of FpvA-PaA in vitro. We showed that the FpvA-PaA complex adopts two different conformations depending on how it was prepared (formed in vitro or in vivo prior to purification). The dihydroquinoline moiety of both conformers is fully protonated, or coordinated by protein charged groups, but the polarity of its environment, its solvent accessibility, and its rotational dynamics are much slower when the FpvA-PaA complex is formed in vivo than in vitro. In the presence of Ga(3+) or Al(3+) ions, the solvent accessibility and mobility of the dihydroquinoline moiety in the two FpvA-PaA complexes are intermediate between those observed for the metal-free ones. In addition, the F?rster resonance energy transfer kinetics from FpvA tryptophan residues to the PaA chromophore differs from one complex to the other, revealing differences in one or more of the donor-acceptor topologies.     

Kinetic effect of some aliphatic amines on yeast alcohol dehydrogenase.

Initial rate studies of ethanol oxidation catalyzed by yeast alcohol dehydrogenase (EC 1.1.1.1) were carried out in the presence of varying concentrations of aliphatic amines over the pH range from 8.0 to 10.5. Aliphatic amines either activate or inhibit the enzyme depending on whether the pH is greater or less than 9.5 suggesting that the protonated amines activate and the nonprotonated amines inhibit the enzyme. Aliphatic amines activate yeast alcohol dehydrogenase by decreasing Kb while they inhibit the enzyme by increasing both Ka and Kia. When both protonated and nonprotonated amines are present in solution, either overall activation or inhibition will be observed depending on the relative concentration of the two amine species.     

Protonation and quaternization of 1, N6-ethenoadenosine: What are the species responsible for the fluorescence of 1, N6-ethenoadenosine?

Methylation of 1,N⁶-ethenoadenosine (εAdo) gives a mixture of N1- and N9-quaternized methyl-3-β-D -ribofuranosylimidazo[2,1-i] purinium salts (m¹εAdo⁺ and m⁹εAdo⁺, respectively). The ratio of the two forms of the protonated εAdo [H¹εAdo⁺]/[H⁹εAdo⁺] has been estimated to be approximately 0.10 by comparing the uv absorption spectra of the protonated species of εAdo and the two nontautomerizable model compounds. In relation to a study on the protonation effect on the fluorescence of εAdo, we have now determined the effect of quaternization on the fluorescence spectra at 293 and 77 K. We have found that m¹εAdo⁺ and m⁹εAdo⁺ are both fluorescent, and the high degree of coincidence between the fluorescence spectra of εAdo and m¹εAdo⁺ at pH 7 is noted. The m¹εAdo⁺ singlet form is a more efficient fluorescer than the m⁹εAdo⁺ ion at room temperature (quantum yields of 0.43 and 0.11, respectively). All the results which are presented in this paper are consistent with the picture that there exist more than one species responsible for the fluorescence of εAdo, depending on the environment of the molecule in aqueous solution (temperature and pH of solvent).     

Solid-state IR-LD spectroscopic and theoretical analysis of glycine-containing peptides and their hydrochlorides

As part of an investigation on the coordination ability of peptides, structural analyses of the solid di-, tri, and tetrapeptides glycyl-glycine (GG), glycyl-glycyl-glycine (GGG), glycyl-glycyl-glycyl-glycine (GGGG), and their protonated hydrochlorides glycyl-glycine.HCl (GGH), glycyl-glycyl-glycine.HCl (GGGH), and glycyl-glycyl-glycyl-glycine.HCl (GGGGH) have been carried out. The quantum chemical calculations (Hartree-Fock/6-31++G**) and linear-dichroic infrared (IR-LD) spectroscopy predict a near to linear structure of the pure ligands, but the experimental IR-LD data are in accordance with a cross-linked disposition of amide fragments in the protonated forms.     

Conformations of DNA strands containing GAGT, GACA, or GAGC tetranucleotide repeats

The (GA)(n) microsatellite has been known from previous studies to adopt unusual, ordered, cooperatively melting secondary structures in neutral aqueous solutions containing physiological concentrations of salts, at acid pH values or in aqueous ethanol solutions. To find more about the primary structure specificity of these structures, we performed parallel comparative studies of related tetranucleotide repeats (GAGC)(5), (GAGT)(5), and (GACA)(5). The general conclusion following from these comparative studies is that the primary structure specificity is fairly high, indicating that not only guanines but also adenines play a significant role in the stabilization of these unusual structures. (GAGC)(5) is a hairpin or a duplex depending on DNA concentration. Neither acid pH nor ionic strength or the presence of ethanol changed the secondary structure of (GAGC)(5) in a significant way. (GACA)(5) forms a weakly stable hairpin in neutral aqueous solutions but forms a duplex at acid pH where cytosine is protonated. (GAGT)(5) behaves most similar to (GAGA)(5). Salt induces its hairpin to duplex transition at neutral pH and an isomerization into another, probably parallel stranded, duplex takes place at acid pH. (GAGT)(5) is the only of the three present 20-mers that responds to ethanol like (GAGA)(5).     

Demonstration of three protonated forms of poly(A): potentiometric and conductometric study of isoionic solutions

It is shown that there are three parts on the potentiometric titration curves of isoionic solutions of poly(A) ascribed to the three protonated structures. Double-helical protonated structures are especially stable in isoionic solution. These parts on potentiometric curves are attributed to the single-stranded poly(A), to the completely protonated double-stranded poly(A+).poly(A+), and to the semiprotonated poly(A+).poly(A) structures: D, A, B forms of poly(A), respectively. pK0 values of these forms are calculated. The D form portion is found to be about 18% in isoionic solution, 40% in KCl solution (from 0.01 to 1.0 M), 40% in solution, containing 1.2 X 10(-3) M MgCl2 and 70% in 8 X 10(-4) M MgCl2 solution. The increase of MgCl2 concentration up to 8 X 10(-4) M leads to complete degradation of the double-helical structure. Only single-stranded D form exists in 5 X 10(-3) M MgCl2 solution. About 5-7% of all protons become inaccessible for titration in all solutions containing KCl and in the presence of small amounts of MgCl2. This phenomenon can not be explained by aggregation of poly(A), because all protons become accessible for titration in more concentrated MgCl2 solution when aggregation of poly(A) is significant and accompanied by the precipitation of sediment insoluble in NaOH. The supposition is made, that unprotonated double-stranded poly(A) can exist in salt-free solution at neutral pH. It is this form that is protonated with decrease of pH.     

Systematic characterisation and seed oil analysis in candidate plus trees of biodiesel plant, Pongamia pinnata

Candidate plus trees (CPTs) of Pongamia pinnata , a potential biodiesel plant occurring across 10 locations in North Guwahati, were identified based on morphological markers (vegetative and reproductive) using combined analysis over locations. Identified CPTs were then multiplied using seed propagation technique in a nursery bed. The performance of the candidate trees with respect to seed and pod traits, the two most important characters with regard to oil, were evaluated using CROPSTAT software for inferring potential genotypes that can be included in programmes aimed at genetic improvement of the species. Total oil content from the seeds of plus trees was also analysed using solvent extraction procedure at their boiling points. Hexane extraction yielded maximum oil content from seeds (33%) compared with petroleum ether (30%). When the seed to solvent ratio varied, no significant difference was noticed on the total oil yield for an individual tree, although the recovery of solvent and the time taken for oil extraction were significantly reduced at higher ratios of solvent used.     

Photo-induced protonation/deprotonation in the GFP-like fluorescent protein Dronpa: mechanism responsible for the reversible photoswitching.

Recently, reversible photoswitching in bulk samples or in individual molecules of Dronpa, a mutant of a green fluorescent protein (GFP)-like fluorescent protein, has been demonstrated. Intense irradiation at 488 nm changed Dronpa in a dim protonated form, and weak irradiation at 405 nm restored it to the bright deprotonated form. Here, we report on the mechanism of photoswitching of Dronpa by means of ensemble and single-molecule spectroscopy. Ensemble spectroscopy shows that the photoswitching can be described, in first approximation, by a three-state model including a deprotonated (B), a protonated (A1), and a photoswitched protonated (A2) forms of the chromophore. While the B and the A1 forms are in a ground state acid-base equilibrium, the B and the A2 forms are reversibly photoswitched upon irradiation with 488 and 405 nm light. At the single-molecule level, the on-times in fluorescence intensity trajectories excited at 488 nm decrease with increasing the excitation power, consistent with the photoswitching from the B to A2 form. The on-times agree well with expected values, which are calculated based on the ensemble spectroscopic properties of Dronpa. The fluorescence trajectory obtained with simultaneous dual-color excitation at 488 and 405 nm demonstrates reversible photoswitching between the B and the A2 forms at the single-molecule level. The efficiency of the photoswitching from the A2 to B form increased with increasing the excitation power of the 405 nm light. Our results demonstrate that Dronpa holds its outstanding photoswitching properties, based on a photo-induced protonation/deprotonation process, even at the single-molecule level.     

Microspectrofluorometry of the protonation state of ellipticine, an antitumor alkaloid, in single cells.

The protonation state and intracellular distribution of ellipticine were investigated in single human mammary T47D cells by confocal laser microspectrofluorimetry. In the cell nucleus, only the protonated form of ellipticine was detected as a direct consequence of its apparent pK increase upon DNA binding. Both protonated and neutral forms were present in the aqueous cytoplasm, where the pH is close to the drug pK. When cells were incubated in high concentrations of K+, a condition that depolarizes the plasma membrane potential, ellipticine cellular accumulation was reduced. In the cytoplasm, ellipticine was mainly bound to mitochondria, and its protonation equilibrium was shifted toward the neutral form. The fluorescence spectrum of ellipticine bound to mitochondria was insensitive to valinomycin, whereas it was markedly shifted toward the protonated form after carbonyl cyanide p-trifluoromethoxy-phenylhydrazone or nigericin addition. Similar studies with ellipticine bound to isolated mitochondria suggest that it behaves as a fluorescent probe of mitochondrial pH in both isolated mitochondria and single living cells.     

NMR studies of the metal-loading kinetics and acid-base chemistry of DOTA and butylamide-DOTA.

The conjugation of a chelating agent to a protein via a covalent linkage has been previously reported to change the metal-binding characteristics of the chelator. A fundamental understanding of these binding changes would enable design of a new generation of metal-chelating agents for biological applications. To assess the effect of conjugation on the commonly used chelating agent 1 4,7, 10-tetraaazacyclododecane-N,N',N',N'-tetraaacetic acid (DOTA), we synthesized a model protein conjugate, 1,4, 7-tris(carboxymethyl)-10-(butylaminocarboxymethyl)-1,4,7, 10-tetraaazacyclododecane (BD) and explored the metal-binding characteristics via NMR. The extent of ionization of the carboxylic acid groups and the two protonated macrocycle nitrogens of DOTA and BD were determined as a function of pH by chemical shift changes in proximal carbon-bonded protons. In addition to the expected sensitivity of the chemical shifts to titration of proximate acidic groups, BD resonances from carbon-bonded protons 10-17 bonds distant from the deprotonation site also shifted significantly, indicating the presence of conformational changes. Furthermore, increased shielding of the amide and alkyl proton signals upon deprotonation of the carboxylic acid groups indicates the presence of pH-dependent hydrogen-bonded BD isoforms. On the basis of these NMR data, we propose new structures for the doubly protonated forms of DOTA and BD. To measure metal loading, the yttrium-loading rates (type I to type II) of DOTA and BD were determined by following the intensity of type I and type II proton signals as a function of time. The yttrium-loading rates of BD are approximately one-half those of DOTA at pHs between 4.6 and 6.5 and 37 degrees C. The loading rates measured as a function of pH indicate that while both the doubly protonated and singly protonated forms of DOTA are reactive to metal loading, only the singly protonated form of BD is reactive.     

Phosphofructokinase. II. Role of ligands in pH-dependent structural changes of the rabbit muscle enzyme.

The effect of ligands, including substrates and allosteric effectors, on the pH-dependent inactivation and reactivation of rabbit muscle phosphofructokinase has been examined in terms of the mechanism proposed previously (Bock, P.E. and Fireden, C. (1976) J. Biol. Chem. 251, 5630-5636). It is concluded thatt many ligands exert their effect by binding preferentially to either protonated or unprotonated forms of the enzyme and thus shifting an apparent pK for the inactivation or reactivation process. ATP and fructose 6-phosphate influence the apparent pK to different extents and in different directions, with ATP binding preferentially to the protonated forms and fructose 6-phosphate to the unprotonated forms. Enzyme inactivated by ATP can be reactivated by the addition of fructose 6-phosphate. The experiments indicate that inactivation and reactivation in the presence of these ligands can occur by kinetically different pathways as has been found for these processes in the absence of ligands. The results are discussed in relation to what might be expected for ligand binding properties of the enzyme as a function of pH, temperature, and enzyme concentration. The effect of ATP and MgATP is complex, perhaps representing more than one site of binding. Citrate appears to bind preferentially to protonated forms of the enzyme while fructose 1,6-bisphosphate and AMP bind preferentially to the unprotonated forms. ADP, K+, and NH4+ appear to have little or no preference in binding to different enzyme forms.     

Theoretical evaluation of isotopic fractionation factors in oxidation reactions of benzene,phenol and chlorophenols

We have studied theoretically the rate determining steps of reactions of benzene with permanganate, perchlorate, ozone and dioxygen in the gas phase and aqueous solution as well as phenol and dichlorophenol in protonated and unprotonated forms in aqueous solution. Kinetic isotope effects were then calculated for all carbon atoms and based on their values isotopic fractionation factors corresponding to compound specific isotopic analysis have been evaluated. The influence of the oxidant, substituents, environment and protonation on the isotopic fractionation factors has been analyzed.     

Density functional and molecular dynamics simulations of local anesthetics in 0.9% NaCl solution

We have made density functional calculations and molecular dynamics (MD) simulations to investigate the structure and pharmacological action of local anesthetics: tetracaine, procaine and lidocaine. The MD simulations were made in a NPT ensemble, in a 0.9% NaCl solution, on both unprotonated and protonated forms of the molecules. The radial distribution function was used to study solvent effects in different regions of the molecules. Although all three anesthetics have different degrees of hydrophobicity, the amino-terminals were the mostly affected by the protonation yielding hydrophilic regions. The charged amino-esters present hydrophilicity on the ester as well as amine terminals. Cl^?  from the solvent solution forms hydrogen bonds via protonated hydrogen attached to nitrogen, yielding neutral molecules, which could, in principle, penetrate the membranes and loose Cl^?  to act in the protonated form. Density functional theory calculations indicated a change in the electrostatic potential and showed that Cl^?  weakly binds to the amine hydrogen, what suggests it is a favorable interaction and supports the existence of the hydrochloric forms of these local anesthetics.     

Comparative binding studies on the interaction of the indoloquinoline alkaloid cryptolepine with the B and the non-canonical protonated form of DNA: A spectroscopic insight

BackgroundLow pH induced nucleic acid polymorphism and the interaction of naturally occurring small molecules with different polymorphic forms of DNA have been the focus in developing new drugs. Recent studies have revealed that low pH plays an active role in growth and development of cancer cells. Our target is to find whether and how the indoloquinoline alkaloid cryptolepine (CRP) interact with different polymorphic forms of natural DNA, in hope to explore this group of alkaloids as new therapeutics.MethodsMultiple spectroscopic techniques that include UV–visible absorption spectrophotometry, fluorimetry, CD spectroscopy along with thermal melting studies were employed to characterize the interaction between the alkaloid cryptolepine with the B and protonated forms of DNA.Results & conclusionsCryptolepine has been found to interact with either forms of DNA. The nature of binding is non-cooperative in both cases. Data show that the affinity of CRP to B form of DNA is relatively higher than that for the protonated form of DNA. Circular dichroic studies reveal that the alkaloid converts the left handed protonated DNA into bound right handed form. Fluorescence quenching experiments reveal that cryptolepine intercalates within the DNA base pairs. Thermal melting studies show that the alkaloid stabilises the DNA structures.General significanceSuch non-B DNA structures are often present at the ‘mutation hotspots’ that are associated with genetic instability related diseases such as cancer. The ability of cryptolepine to interact to such non-B DNA structures makes it a useful substrate in the designing of potential chemotherapeutic agents.