Reactivities of hydroxylamine and sodium bisulphite with carbonyl-containing haems with the prosthetic groups of the erythrocyte green haemoproteins

The reactivities of alkaline NH(2)OH and neutral NaHSO(3) with carbonyl and olefinic groups conjugated with the tetrapyrrole nucleus of haems were studied. The reactions were carried out with 2-3nmol of haem a, spirographis haem, isospirographis haem, 2,4-diacetyldeuterohaem and protohaem. Vinyl side chains were found to be insensitive to the chemical action of both alkaline NH(2)OH and neutral NaHSO(3). The formyl-containing haems reacted rapidly with both reagents at room temperature, as evidenced by sizable hypsochromic shifts of the reduced pyridine haemochrome spectrum. In less alkaline solution, the reactions of these formyl-containing haems with NH(2)OH were much slower. 2,4-Diacetyldeuterohaem reacted with alkaline NH(2)OH, but not with neutral NaHSO(3). These rapid, simple and straightforward tests are readily usable in differentiating among formyl, acetyl and other electron-withdrawing side chains conjugated with the tetrapyrrole ring of haems. We applied these observations to an investigation of the two unique prosthetic groups of the bovine erythrocyte green haemoproteins. The prosthetic groups of these two proteins were isolated and spectrally characterized. Under the conditions used, the haems did not react with either NH(2)OH or NaHSO(3), but were altered by dithionite, suggesting that the previous interpretation that a formyl group was present [Hultquist, Dean & Reed (1976) J. Biol. Chem.251, 3927-3932] may have been premature. These studies also provide evidence that the alpha-hydroxyfarnesylethyl side chain of haem a affects the alpha-band maximum, but not the beta- or Soret bands of the reduced pyridine haemochrome spectrum of haem a.     

Site-specific mutations provide new insights into the origin of pH effects and alternative spectral forms in the photoactive yellow protein from Halorhodospira halophila

Acid/base titrations of wild-type PYP and mutants, either in buffer or in the presence of chaotropes such as thiocyanate, establish the presence of four spectral forms including the following: a neutral form (446-476 nm), an acidic form (350-355 nm), an alkaline form (430-440 nm), and an intermediate wavelength form (355-400 nm). The acidic species is formed by protonation of the oxyanion of the para-hydroxy-cinnamyl cysteine chromophore as a secondary result of acid denaturation (with pK(a) values of 2.8-5.4) and often results in precipitation of the protein, and in the case of wild-type PYP, eventual hydrolysis of the chromophore thioester bond at pH values below 2. Thus, the large and complex structural changes associated with the acidic species make it a poor model for the long-lived photocycle intermediate, I(2), which undergoes more moderate structural changes. Mutations at E46, which is hydrogen-bonded to the chromophore, have only two spectral forms accessible to them, the neutral and the acidic forms. Thus, an intact E46 carboxyl group is essential for observation of either intermediate or alkaline wavelength forms. The alkaline form is likely to be due to ionization of E46 in the folded protein. We postulate that the intermediate wavelength form is due to a conformational change that allows solvent access to E46 and formation of a hydrogen-bond from a water molecule to the carboxylic acid group, thus weakening its interaction with the chromophore. Increasing solvent access to the intermediate spectral form with denaturant concentration results in a continuously blue-shifted wavelength maximum.     

Molecular dynamics simulation of the unfolding of the human prion protein domain under low pH and high temperature conditions

Four 10-ns molecular dynamics (MD) simulations of the human prion protein domain (HuPrP 125-228) in explicit water solution have been performed. Each of the simulations mimicked a different environment of the protein: the neutral pH environment was simulated with all histidine residues neutral and bearing a ND proton and with other titratable side chains charged, the weakly acidic environment was simulated with all titratable side chains charged, the strongly acidic environment was simulated with all titratable side chains protonated. The protein in neutral pH environment was simulated at both ambient (298 K) and higher (350 K) temperatures. The native fold is stable in the neutral pH/ambient temperature simulation. Through out all other simulations, a quite stable core consisted of 10-20 residues around the disulfide bond retain their initial conformations. However, the secondary structures of the protein show changes of various degrees compared to the native fold, parts of the helices unfolded and the beta-sheets extended. Our simulations indicated that the heat-induced unfolding and acid-induced unfolding of HuPrP might follow different pathways: the initial stage of the acid-induced unfolding may include not only changes in secondary structures, but also changes in the tertiary structures. Under the strongly acidic condition, obvious tertiary structure changes take place after 10-ns simulation, the secondary structure elements and the loops becoming more parallel to each other, resulting in a compact state, which was stabilized by a large number of new, non-native side chain-side chain contacts. Such tertiary structure changes were not observed in the higher temperature simulation, and intuitively, they may favor the further extension of the beta-sheets and eventually the agglomeration of multiple protein molecules. The driving forces for this tertiary structure changes are discussed. Two additional 10-ns MD simulations, one with Asp202 protonated and the other with Glu196 protonated compared to the neutral pH simulation, were carried out. The results showed that the stability of the native fold is very subtle and can be strongly disturbed by eliminating a single negative charge at one of such key sites. Correlations of our results with previous experimental and theoretical studies are discussed.     

Resonance-stabilized phenylazo-ene-phenylimine cations of cyclobutanetetraone derivatives

Cyclobutenedione phenylazo-phenylamines were found to exhibit bathochromic shifts in acidic media and hypsochromic shifts in basic media, like phenylazo-phenylhydrazones. The bathochromic shifts are due to the formation of resonance-stabilized cations and the hypsochromic shifts to enolization. The phenylazo-phenylamines and their cations and anions have been studied by NMR spectroscopy.     

Effects of pH on murine insulinoma betaTC3 cells

Confluent monolayer cultures of betaTC3 cells were exposed for 4 h to acidic, neutral, or alkaline pH media. Studies determined the impact of pH on viability, insulin secretion rate, glucose consumption rate, lactate production rate, and ATP content. Cell viability was not affected by exposure to media of different pH (>95% for all groups). Insulin release from cells exposed to acidic media (pH of 6.4) was approximately 75% higher than that from cells exposed to either neutral (pH of 7.1) or alkaline (pH of 7.8) conditions. Conversely, ATP content was significantly reduced in cultures exposed to acidic conditions, although there was no statistical difference between neutral and alkaline conditions. Glucose consumption and lactate production rates increased linearly with increasing pH.     

FTIR spectra of solid poly-l-lysine in the stretching NH mode range

Three bands at 3270 cm(-1), 3200 cm(-1) and 3030 cm(-1) are found in the IR stretching proton (nu(1)) mode spectral range in spectra of solid poly-l-lysine (PLL). Strong quantitative changes of these bands are observed in samples dried from water solutions with different pH. The narrow band at 3270 cm(-1), which is strong in the spectrum of PLL precipitated from pH=12 alkaline medium, is assigned to the nu(1) peptide proton mode of NH-CO (amide A) of the beta-sheet structure type. The band at 3200 cm(-1), which is intensified in PLL precipitated from pH=1 acidic medium, relates to the nu(1) peptide mode in the random coil structure. The band at 3030 cm(-1), whose peak intensity increases two-fold in going from alkaline to acidic medium, is assigned to the nu(1) modes of protonated NH(3)(+) side chain groups. The frequencies of all bands were used for estimating H-bond energy relying on an empirical correlation between this property and the red shift of the nu(1) band. The enthalpy of the secondary structure transition from beta-sheet to the random coil, which is observed in PLL at the change of pH from 11 to 1 amounts to 4.7 kJ mol(-1).     

Interaction and inhibitory effect of ammonium cation in the oxygen evolving center of photosystem II

Photosynthetic O(2) evolution takes place at the Mn cluster in photosystem II (PSII) by oxidation of water. It has been proposed that ammonia, one of water analogues, functions as an inhibitor of O(2) evolution at alkaline pH. However, the detailed mechanism of inhibition has not been understood yet. In this study, we investigated the mechanism of ammonia inhibition by examining the NH(4)Cl-induced inhibition of O(2) evolution in a wide pH range (pH 5.0-8.0) and by detecting the interaction site using Fourier transform infrared (FTIR) spectroscopy. In addition to intact PSII membranes from spinach, PSII membranes depleted of the PsbP and PsbQ extrinsic proteins were used as samples to avoid the effect of the release of these proteins by salt treatments. In both types of samples, oxygen evolution activity decreased by approximately 40% by addition of 100 mM NH(4)Cl in the range of pH 5.0-8.0. The presence of inhibition at acidic pH without significant pH dependence strongly suggests that NH(4)(+) cation functions as a major inhibitor in the acidic pH region, where neutral NH(3) scarcely exists in the buffer. The NH(4)Cl treatment at pH 6.5 and 5.5 induced prominent changes in the COO(-) stretching regions in FTIR difference spectra upon the S(1) → S(2) transition measured at 283 K. The NH(4)Cl concentration dependence of the amplitude of the spectral changes showed a good correlation with that of the inhibition of O(2) evolution. From this observation, it is proposed that NH(4)(+) cation interacts with carboxylate groups coupled to the Mn cluster as direct ligands or proton transfer mediators, causing inhibition of the O(2) evolving reaction.     

Ammonia/potassium exchange in methanogenic bacteria

Methanospirillum hungatei exposed to ammonia in a K+-free buffer lost up to 98% of the cytoplasmic K+ through an ammonia/K+ exchange reaction. The exchange was immediate, and occurred in cells poisoned by air or by other metabolic inhibitors. Additions of NH4OH or various NH+4 salts (or methylamine) were most effective in causing K+ depletion in media of alkaline pH, suggesting that NH3 was the chemical species crossing the membrane. In alkaline media, the exchange reaction resulted in a dissipation of the transmembrane pH gradient (inside acidic), but had only small effects on the membrane potential until concentrations of ammonia were used above those required to abolish the K+ gradient. Through the use of NH4Cl to vary the cytoplasmic pH at a constant acidic external pH, and NH4OH to abolish the transmembrane pH gradient at various alkaline external pH values, we conclude that methanogenesis is sensitive to both the pH of the cytoplasm and the medium. Methanogenesis in Msp. hungatei and Methanosarcina barkeri was inhibited dramatically at external pH values more acidic than 6.5 or more alkaline than 7.5. Dramatic K+ depletion in response to ammonia additions at pH 8.0 occurred with Ms. barkeri, another strain of Msp. hungatei, Escherichia coli, and Bacillus polymyxa. In several other methanogens, ammonia/potassium exchange was hardly detected.     

Anthocyanin from strawberry (Fragaria ananassa) with the novel aglycone, 5-carboxypyranopelargonidin

An anthocyanin, 1, with the novel 4-substituted aglycone, 5-carboxypyranopelargonidin, was isolated in small amounts from the acidified, methanolic extract of strawberries, Fragaria ananassa Duch., by preparative HPLC after purification by partition against ethyl acetate, Amberlite XAD-7 and Sephadex LH-20 column chromatography. It was identified mainly by 2D NMR spectroscopy and electrospray LC-MS as the 3-O-beta-glucopyranoside of 5-carboxy-2-(4-hydroxyphenyl)-3,8-dihydroxy-pyrano[4,3,2-de]-1-benzopyrylium, an anthocyanidin which is homologous to 5-carboxypyranomalvidin (vitisidin A) reported in red wines and 5-carboxypyranocyanidin recently isolated from red onions. By comparison of UV-Vis absorption spectra, 1 showed in contrast to 2, pelargonidin 3-O-beta-glucopyranoside, a local absorption peak around 360 nm, a hypsochromic shift (8 nm) of the visible absorption maximum, and lack of a distinct UV absorption peak around 280 nm. The similarities between the absorption spectra of 1 in various acidic and neutral buffer solutions implied restricted formation of the instable colourless equilibrium forms, which are typical for most anthocyanins in weakly acidic solutions. The molar absorptivity (epsilon) of 1 varied little with pH contrary to similar values of for instance the major anthocyanin in strawberry, 2. However, 2 revealed higher epsilon-values than 1 at all pH values except 5.1. At pH 5.1, the epsilon-value of 1 (6250) was nearly four times the corresponding value of 2 (1720), which showed the potential of 5-carboxypyranopelargonidin derivatives as colorants in solutions with pH around 5. The colours of 1 and 2 in buffered solutions with pH 1.1 and pH 6.9 have been described by the CIELAB coordinates h(ab) (hue angle), C* (chroma), and L* (lightness).     

Colony formations in a halotolerantBrevibacterium sp. JCM 6894 on solid medium with different pH values

Viable cells of a halotolerantBrevibacterium sp. JCM 6894 grown in a liquid medium with pH 7.1 were enumerated as the colony-forming cells on three kinds of agar media with different pH values. Unexpectedly they were lower at neutral pH rather than acidic or alkaline pH. This tendency was invariable regardless of the changes in the concentrations of nutrients in the agar medium as well as in the growth phases of the cells. From the comparison of cell growth between liquid and solid media with different pHs, we notified the importance of the pH changes in liquid medium accompanied with growth. Effects of salts and pH of the liquid medium on protonmotive force (Δp) was estimated from membrane potentials (ΔΨ) and proton gradients (ΔpH) of the strain JCM 6894. In the absence of salts, Δp of the strain JCM 6894 was the largest at neutral pH, which was conflicting with the result of cell viability. The addition of NaCl led to the reduction of Δp at acidic pH, mainly due to the dissipation of ΔΨ, which seems to be consistent with the lower numbers of colony formed at acidic pH in the presence of NaCl.     

Synthesis, characterization and anticonvulsant activity of enaminones. Part 6: Synthesis of substituted vinylic benzamides as potential anticonvulsants

A comparison of enaminones from various unsubstituted and p-substituted benzamides to the analogous benzylamines has been undertaken with the aim of elucidating the essential structural parameters necessary for anticonvulsant activity. Initial studies on methyl 4-N-(benzylamino)-6-methyl-2-oxocyclohex-3-en-1-oate, 3a, 3-N-(benzylamino)cyclohex-2-en-1-one, 3p, and 5,5-dimethyl-3-N-(benzylamino)-cyclohex-2-en-1-one, 3r indicated that benzylamines possessed significant anti-maximal electroshock seizure (MES) activity. Evaluation of the analogous benzamides revealed significant differences in anticonvulsant activity, these differences were most probably related to the differences in their three-dimensional structures.     

Pyridoxal 5'-phosphate: a possible physiological substrate for alkaline phosphatase in human neutrophils

The intracellular localization of pyridoxal phosphatase activity was demonstrated in human neutrophils by electron microscope cytochemistry. Under alkaline conditions, an enzyme active against pyridoxal phosphate was localized to a cytoplasmic granule population, the phosphasome. These granules have previously been shown by electron microscope cytochemical techniques and by subcellular fractionation to be rich in alkaline phosphatase. Under acidic conditions, a phosphatase activity against pyridoxal phosphate was localized to intracellular multilamellar bodies resembling secondary lysosomes. These were quite distinct from the primary, secondary and phosphasome granules and this unique localization corresponds to that previously demonstrated (tertiary granules) by subcellular fractionation studies of these cells. The similarity in the enzyme reaction requirements of alkaline pyridoxal phosphatase and alkaline phosphatase, and their localization to the same subcellular organelle, suggests that pyridoxal phosphate may be a physiological substrate for human neutrophil alkaline phosphatase.     

Preparative binding of Coomassie brilliant blue to bovine serum

Laboratory scale preparation of bovine serum albumin (BSA) stained with Coomassie brilliant blue (CBB) at alkaline pH is first described. Physical-chemical analyses of CBB-BSA showed that the unprotonated (anion) CBB dye binds tightly to BSA in buffered media of pH 8.2. Characteristic differences in spectra lambda(max) and molar absorptivities were found for the free anion CBB dye versus the CBB-BSA complex. Binding studies with low versus high dye/protein concentration ratios at alkaline pH gave values for n, binding site numbers, and K, intrinsic binding coefficient, consistent with those reported in analytical studies under acidic pH, but higher than values for neutral pH. Comparative analyses of Beer's law plots for the alkaline CBB-BSA complex under different experimental conditions showed its high stability toward various interferences, such as pH, strong detergents, temperature, light, prolonged storage, as well as high affinity for tannins. The hydrophobic nature of the CBB-BSA association at alkaline pH was tested.     

High-resolution nuclear magnetic resonance determination of transfer RNA tertiary base pairs in solution. 1. Species containing a small variable loop.

Eight class I tRNA species have been purified to homogeneity and their proton nuclear magnetic resonance (NMR) spectra in the low-field region (-11 to -15 ppm) have been studied at 360 MHz. The low-field spectra contain only one low-field resonance from each base pair (the ring NH hydrogen bond) and hence directly monitor the number of long-lived secondary and tertiary base pairs in solution. The tRNA species were chosen on the basis of their sequence homology with yeast phenylalanine tRNA in the regions which form tertiary base pairs in the crystal structure of this tRNA. All of the spectra show 26 or 27 low-field resonances approximately 7 of which are derived from tertiary base pairs. These results are contrary to previous claims that the NMR spectra indicate the presence of resonances from secondary base pairs only, as well as more recent claims of only 1-3 tertiary resonances, but are in good agreement with the number of tertiary base pairs expected in solution based on the crystal structure. The tertiary base pair resonances are stable up to at least 46 degrees C. Removal of magnesium ions causes structural changes in the tRNA but does not result in the loss of any secondary or tertiary base pairs.     

Proton nuclear magnetic resonance study of the effect of pH on tRNA structure.

The low-field 220-MHz proton nuclear magnetic resonance (NMR) spectra of four tRNA molecules, Escherichia coli tRNAPhe, tRNA1Val, and tRNAfMet1, and yeast tRNAPhe, at neutral and mildly acidic pH are compared. We find a net increase in the number of resonances contributing to the -9.9-ppm peak (downfield from sodium 4,4-dimethyl-4-silapentanesulfonate) in three of these tRNAs at pH 6, while tRNAfMet1 does not clearly exhibit this behavior. The increase in intensity at this resonance position is half-completed at pH 6.2 in the case of yeast tRNAPhe. An alteration at the 5'-phosphate terminus is not involved, since removal of the terminal phosphate does not affect the gain in intensity at -9.9 ppm. Based on a survey of the tertiary interactions in the four molecules, assuming that they possess tertiary structures like that of yeast tRNAPhe at neutral pH, we tentatively attribute this altered resonance in E. coli and yeast tRNAPhe to the protonation of the N3 of the adenine residue at position 9 which results in the stabilization of the tertiary triple A23-U12-A9. This intepretation is supported by model studies on the lowfield proton NMR spectrum of AN oligomers at acid pH, which reveal an exchanging proton resonance at -9.4 ppm if the chain length N greater than or equal to 6.     

Maturation of an NH2-terminally extended thermostable alpha-amylase in Bacillus subtilis: a possible mechanism examined by in vitro experiments.

An artificially inserted extra peptide (21 amino acid peptide) between the B. subtilis alpha-amylase signal peptide and the mature thermostable alpha-amylase was completely cleaved by B. subtilis alkaline protease in vitro. The cleavage to form a mature enzyme was observed between pH 7.5 and 10, but not between pH 6.0 and 6.5, although a similar protease activity toward Azocall was observed between pH 6.0 and 7.5. To analyze the effects of pH on the cleavage, CD spectra at pH 6, 8, and 11 of the NH2-terminally extended thermostable alpha-amylase were analyzed and the results were compared with those of the mature form of the alpha-amylase. It is suggested that the cleavage of the NH2-terminally extended peptide is controlled by the secondary and tertiary structure of the precursor enzyme. Similar cleavage of different NH2-terminally extended peptides by the alkaline protease was also found in other hybrid thermostable alpha-amylases obtained.     

Cytoplasmic hydrogen ion diffusion coefficient.

The apparent cytoplasmic proton diffusion coefficient was measured using pH electrodes and samples of cytoplasm extracted from the giant neuron of a marine invertebrate. By suddenly changing the pH at one surface of the sample and recording the relaxation of pH within the sample, an apparent diffusion coefficient of 1.4 +/- 0.5 x 10(-6) cm2/s (N = 7) was measured in the acidic or neutral range of pH (6.0-7.2). This value is approximately 5x lower than the diffusion coefficient of the mobile pH buffers (approximately 8 x 10(-6) cm2/s) and approximately 68x lower than the diffusion coefficient of the hydronium ion (93 x 10(-6) cm2/s). A mobile pH buffer (approximately 15% of the buffering power) and an immobile buffer (approximately 85% of the buffering power) could quantitatively account for the results at acidic or neutral pH. At alkaline pH (8.2-8.6), the apparent proton diffusion coefficient increased to 4.1 +/- 0.8 x 10(-6) cm2/s (N = 7). This larger diffusion coefficient at alkaline pH could be explained quantitatively by the enhanced buffering power of the mobile amino acids. Under the conditions of these experiments, it is unlikely that hydroxide movement influences the apparent hydrogen ion diffusion coefficient.     

Alternative mechanisms of vacuolar acidification in H(+)-ATPase-deficient yeast

Acidification of the endosomal/lysosomal pathway by the vacuolar-type proton translocating ATPase (V-ATPase) is necessary for a variety of essential eukaryotic cellular functions. Nevertheless, yeasts lacking V-ATPase activity (Deltavma) are viable when grown at low pH, suggesting alternative methods of organellar acidification. This was confirmed by directly measuring the vacuolar pH by ratio fluorescence imaging. When Deltavma yeasts were cultured and tested in the acidic conditions required for growth of V-ATPase-deficient mutants, the vacuolar pH was 5.9. Fluid-phase pinocytosis of acidic extracellular medium cannot account for these observations, because the V-ATPase-independent vacuolar acidification was unaffected in mutants deficient in endocytosis. Similarly, internalization of the plasmalemmal H(+)-ATPase (Pma1p) was ruled out, because overexpression of Pma1p failed to complement the Deltavma phenotype and did not potentiate the vacuolar acidification. To test whether weak electrolytes present in the culture medium could ferry acid equivalents to the vacuole, wild-type and the Deltavma yeasts were subjected to sudden changes in extracellular pH. In both cell types, the vacuoles rapidly alkalinized when external pH was raised from 5.5 (the approximate pH of the culture medium) to 7.5 and re-acidified when the yeasts were returned to a medium of pH 5.5. Importantly, these rapid pH changes were only observed when NH(4)(+), routinely added as a nitrogen source, was present. The NH(4)(+)-dependent acidification was not due to efflux of NH(3) from the vacuole, as cells equilibrated to pH 7.5 in the absence of weak electrolytes rapidly acidified when challenged with an acidic medium containing NH(4)(+). These findings suggest that although NH(3) can act as a cell-permeant proton scavenger, NH(4)(+) may function as a protonophore, facilitating equilibration of the pH across the plasma and vacuolar membranes of yeast. The high concentration of NH(4)(+) frequently added as a nitrogen source to yeast culture media together with effective NH(4)(+) transporters thereby facilitate vacuolar acidification when cells are suspended in acidic solutions.     

The staining properties of thiazolylazoalkylaminophenol derivatives in histochemical staining of cadmium

In the continuous research for a more sensitive chelator for cadmium, thiazolylazoalkylaminophenol derivatives were synthesized. The staining sensitivity for cadmium and molar absorptivity of their cadmium chelates were examined. Though the molar absorptivity of bromothiazolylazodiethylaminophenol(Br-TADA)-cadmium chelate demonstrated an order of 10(5), the staining sensitivity of Br-TADA was unexpectedly low. The lowering of the sensitivity of Br-TADA may be attributed to alkaline hydrolysis and a decrease in the stability constant of the cadmium chelate.     

Polyelectrolyte complexes between (cross-linked) N-carboxyethylchitosan and (quaternized) poly[2-(dimethylamino)ethyl methacrylate]: preparation, characterization, and antibacterial properties

Novel polyelectrolyte complexes (PECs) between N-carboxyethylchitosan (CECh) and well-defined (quaternized) poly[2-(dimethylamino)ethyl methacrylate] (PDMAEMA) have been obtained. The modification of chitosan into CECh allows the preparation of PECs in a pH range in which chitosan cannot form complexes. The CECh/PDMAEMA complex is formed in a narrow pH range around 7. The quaternization of the tertiary amino groups of PDMAEMA enables complex formation with CECh both in neutral and in alkaline medium. Cross-linked CECh is also capable of forming complexes with (quaternized) PDMAEMA. The antibacterial activity of (cross-linked) CECh, (quaternized) PDMAEMA, and their complexes against Escherichia coli has been evaluated. In contrast to (quaternized) PDMAEMA, (cross-linked) CECh exhibits no antibacterial activity. The complex formation between cross-linked CECh and (quaternized) PDMAEMA results in a loss of the inherent antibacterial activity of the latter in neutral medium. In acidic medium, the complexes exhibit strong antibacterial activity due to complex disintegration and release of (quaternized) PDMAEMA.