Molecular Design of Trypsin Towards High Substrate Selectivity

Molecular design of trypsin mutants towards higher substrate specificity for arginine or ly-sine type substrates was studied. The difference in side chain pKa of arginine and lysine was utilized in redesigning trypsin. If the enzyme could react effectively at a pH higher than lysine's pKa but lower than that of arginine, it would react more selectively with arginine-type substrates, since in that pH range, the side chain of arginie remain protonated, while that of lysine is deprotonated. For trypsin. the change of histidine (57)'s pKa reflects the shift in reaction optimum pH. Electrostatic calculations showed that when surface positive residues were mutated into neutral or negative ones, the pKa of histidine(57) would be raised and those surface charges within a cone of 70 degree around histidine(57) have strong influence on its pKa. Several sites were suggested in rat trypsin which might serve as potential mutation locations to make trypsin active at a higher pH, thus more selective towards arginine t     

Monolayer studies of single-chain polyprenyl phosphates

The monolayer properties of some single-chain polyprenyl phosphates (phytanyl, phytyl, and geranylgeranyl phosphates), which we regard as hypothetical primitive membrane lipids, were investigated at the air-water interface by surface pressure-area (pi-A) isotherm measurements. The molecular area/ pressure at various pH conditions dependence revealed the acid dissociation constants (pKa values) of the phosphate. The pKa values thus obtained at the air-water interface (pKa1 = 7.1 and pKa2 = 9.4 for phytanyl phosphate) were significantly shifted to higher pH than those observed in the bilayer state in water (pKa1 = 2.9 and pKa2 = 7.8). The difference in pKa values leads to a stability of the phosphate as both monolayer and bilayer states in a pH range of 2-6. In addition, the presence of ions such as sodium, magnesium, calcium, and lanthanum in the subphase significantly altered the stability of the polyprenyl phosphate monolayers, as shown by the determination of monolayer collapse and compression/expansion hysteresis. Although sodium ions in the subphase showed only a weak effect on the stabilization of the monolayer, addition of magnesium ions or of a small amount of calcium ions significantly suppressed the dissolution of the monolayer into the subphase and increased its mechanical stability against collapse. In contrast, the presence of larger amounts of calcium or of lanthanum ions induced collapse of the monolayers. Based on these experimental facts, a plausible scenario for the formation of primitive cell membrane by transformation of a monolayer to vesicle structures is proposed.     

pH值对亮黄水溶液及CTAB胶束溶液共振Raman光谱的影响

我们曾研究了黑皂膜的界面性质,为深入了解pH对偶氮苯类化合物在水溶液中及胶束表面上结构变化的影响,本文选用亮黄(BY)为谱学探针,仔细考察了其水溶液及溴化十六烷基三甲铵(CTAB)胶束溶液的共振Raman光谱,根据谱带指认,描述了由酸碱性导致的BY分子的结构变化,其中包括共振体间平衡和酸碱平衡。     

pH effect on cysteine and cystine behaviour at hanging mercury drop electrode

The pH effects on the electrochemical reactions of thiol and disulphide groups on mercury electrodes have been studied. These groups facilitate the oxidation of mercury from the electrode and its conversion into mercury thiolates. Under the appropriate experimental conditions, these thiolates form a compact film around the electrode. The formation of this film can be detected by the appearance of a spike current by cyclic voltammetry. Adsorption at the mercury surface of these groups is conditioned by the charge of the molecule, which in turn, is a consequence of the pH. Therefore, in cysteine solution, the compact film appears only when pH lies between the pKa1 and pKa2, and between the pKa2 and pKa3 in cystine solutions. At these pH values cysteine and cystine carry a zero net charge.     

Titration of ionizable monolayers by measurement of the electric double-layer force

We have determined the pKa of surface-bound primary amine groups by determining the surface potential as a function of solution pH from the magnitude of the electric double-layer force. Using colloid-probe atomic force microscopy (AFM), we measured the force as a function of separation between a particle of radius R = 10 microm and a planar surface, each coated with a self-assembled monolayer of HS(CH2)2CONH((CH2)2O)8(CH2)2NH2. The force was measured from pH 3 to 7, and the surface potential was determined by fitting the results to solutions of the nonlinear Poisson-Boltzmann equation. The surface pKa of the primary amine group was found to be 5.0 +/- 0.2, in agreement with the results of contact-angle and chemical-force titrations on similar surfaces with primary amine groups. The surface charge density indicates that less than 1% of the NH2 groups are dissociated at pH 3, suggesting that ionization is very unfavorable in the local environment of the ethylene oxide chains. This noncontact method should be of general applicability to surfaces with ionizable groups and avoids the possible complications of large contact forces on the surfaces under study.     

Surface complexation mechanism and modeling in Cr(III) biosorption by a microalgal isolate, Chlorella miniata

The mechanism involved in the removal of Cr(III) by a green microalgal isolate, Chlorella miniata, was examined based on a series of batch experiments and microscopic analyses, and a mathematical model was proposed. Results showed that Cr(III) biosorption increased with the increase of pH from 2.0 to 4.5, and no significant changes in biosorption outside this pH range. Langmuir isotherm indicated that the maximum Cr(III) sorption capacity of Chlorella miniata was 14.17, 28.72, and 41.12 mg g(-1) biomass at pH 3.0, 4.0, and 4.5, respectively. Results from desorption studies, SEM (scanning electron microscopy), TEM (transmission electron microscopy), and EDX (energy-dispersive X-ray spectroscope) analyses confirmed that surface complexation was the main process involved in Cr(III) biosorption. Potentiometric titration revealed that carboxyl (pKa1 = 4.10), phosphonate (pKa2 = 6.36) and amine (pKa3 = 8.47) functional groups on the surface of Chlorella miniata were the possible sites for Cr uptake, and their average amounts were 0.53, 0.39, and 0.36 mmol g(-1) biomass, respectively. A surface complexation model further indicated that carboxyl group played the main role in Cr(III) complexation, with a binding constant of K11 = 1.87 x 10(-4) and K12 = 6.11 x 10(-4) for Cr3+ and Cr(OH)2+, respectively. This model also suggested that the hydroxy species was more easily to complex with the cell surface of Chlorella miniata.     

Redox and acid-base coupling in ultrathin polyelectrolyte films

A single layer of poly(allylamine) with a covalently attached osmium pyridine-bipyridine complex adsorbed onto a Au surface modified by mercaptopropanesulfonate has been studied theoretically with a molecular approach and experimentally by cyclic voltammetry. These investigations have been carried out at different pHs and ionic strengths of the electrolyte solution in contact with the redox polyelectrolyte modified electrode. The theory predicts strong coupling between the acid-base and redox equilibria, particularly for low ionic strength, pH close to the pKa, and high concentration of redox sites. The coupling leads to a decrease in the peak potential at pH values above the apparent pKa of the weak polyelectrolyte, in good agreement with the experimental pH dependence at 4 mM NaNO3. Theoretical calculations suggest that the inflection point in the peak position versus pH curves can be used to estimate the apparent pKa of the amino groups in the polymer. Comparison of the apparent pKa for PAH-Os in the film with that of poly(allylamine) reported in the literature shows that the underlying charged thiol strongly influences charge regulation in the film. A systematic study of the film thickness and the degree of protonation in sulfonate and amino groups for solutions of different pH and ionic strength shows the coupling between the different interactions. It is found that the variation of the film properties has a non-monotonic dependence on bulk pH and salt concentration. For example, the film thickness shows a maximum with electrolyte ionic strength, whose origin is attributed to the balance between electrostatic amino-amino repulsions and amino-sulfonate attractions.     

Ruthenium(II) complex of Hbopip: synthesis, characterization, pH-induced luminescence "off-on-off" switch, and avid binding to DNA

A novel ruthenium(II) complex of [Ru(bpy)2(Hbopip)](ClO4)2 (in which bpy=2,2'-bipyridine, Hbopip=2-(4-benzoxazolyl)phenylimidazo[4,5-f][1,10]phenanthroline) was synthesized and characterized. The spectrophotometric pH titrations of the complex showed that it acted as a pH-induced luminescence "off-on-off" switch: a luminescence off-on switch with a luminescence enhancement factor of IpH=3.0/IpH=1.0=20 occurring over a narrow pH range of 1.00-3.00 plus a luminescence on-off switch with a luminescence enhancement factor of 3 over a pH range of 3.20-9.40. The excited-state ionization constant of the complex derived, pKa1*=3.06, is 1.36 pKa units greater than the ground-state pKa1=1.70, and pKa2*=5.01 and pKa3*=8.22 are comparable to the ground-state pKa2=5.23 and pKa3=8.22, respectively. The complex avidly bound to calf thymus DNA with a large binding constant of (1.2+/-0.3)x10(7) M-1 in buffered 50 mM NaCl, as evidenced by UV-vis and luminescence titrations, steady-state emission quenching by [Fe(CN)6]4-, DNA competitive binding with ethidium bromide, viscosity measurements, and DNA melting experiments.     

Acid-base properties and zeta potentials of self-assembled monolayers obtained via in situ transformations

Siloxane-anchored, self-assembled monolayers (SAMs) on single crystal Si were prepared with a variety of surface functional groups using a single commercially available surfactant (1-bromo-11-(trichlorosilyl)undecane) followed by in situ transformations. Polar (thioacetate and thiol), nonpolar (methyl), acidic (sulfonic and carboxylic), basic (various amines), and ionic (alkylammonium) surface functionalities were prepared. For primary amine and sulfonate surfaces, the degree of surface charge as a function of pH was determined ex situ using X-ray photoelectron spectroscopy (XPS). Sulfonate SAMs exhibited much higher effective pKa (approximately 2) than dilute sulfonic acid (-5 to -6), and amine SAMs exhibited much lower pKa (approximately 3) than dilute organic amines (approximately 10). This is attributed to the stabilization of nonionized groups by adjacent ionized groups in the SAM. Zeta potentials of these SAMs as a function of pH were consistent with the XPS results and indicated that ionizable SAM surfaces can generate surface potentials much higher than those of nonionic SAMs (thioacetate, methyl) and typical oxide surfaces.     

pH gradient high-performance liquid chromatography: theory and applications

pH gradient high-performance liquid chromatography (HPLC) is a method of reversed-phase high-performance liquid chromatography suitable for ionogenic substances. It consists in programmed increase during the chromatographic process of the eluting strength of eluent with respect to the analytes separated. On the analogy of the conventional organic modifier gradient reversed-phase HPLC, in the pH gradient approach the eluting strength of the mobile phase increases due to its changing pH: increasing in case of acids or decreasing in case of bases. At the same time the content of organic modifier remains constant. A theory of the pH gradient HPLC has been elaborated. The resulting mathematical model is easily manageable. Its ability to predict changes in retention and separation of analytes following the changes in chromatographic conditions is demonstrated. The pH gradient method is uniquely suitable to determine pKa values of analytes. An equation is presented allowing to calculate pKa values basing on appropriate retention data. The effects on pKa are discussed of the concentration of methanol in the mobile phase. The RP HPLC-derived pKa data correlate to the reference pKa values (w(w)pKa) but are not identical. That may be explained by the effects on the chromatographically determined pKa of the specific interactions of analytes with stationary phases. The proposed pH gradient RP HPLC procedure offers a fast and convenient means to get comparable acidity parameters for larger series of compounds, like drug candidates, also when the analytes are available only in minute amounts and/or as complex mixtures.     

Determination of pKa values of anthraquinone compounds by capillary electrophoresis

In this paper, the dissociation constants (pKa1, pKa2) of five anthraquinones were determined from the relation between the effective mobility at different pH values and the buffer pH value, which was derived from the basic electrophoresis theory and the dissociation equilibrium of a binary acid. In addition, the changes of pKa values of the five compounds were also investigated when organic modifiers were added to the buffer system.     

Charging behavior of the gibbsite basal (001) surface in NaCl solution investigated by AFM colloidal probe technique

The charging behavior of the gibbsite gamma-Al(OH)3 basal (001) surface in aqueous solution is important for correctly modeling the overall charging properties of gibbsite particles which controls surface phenomena such as adsorption and crystal growth. However, the question of whether the hydroxyl groups on the basal plane are proton active has been raised recently both from experimental and theoretical points of view. Using gibbsite crystals prepared from industrial Bayer process, the surface potentials of cleaved (001) surfaces were calculated from forces measured by the colloidal probe technique in 1 mM NaCl solution with differing pH. It was surprisingly found that the basal plane is proton active in pH less than 7 and protonation seems to level off at about pH 5. The potential-pH data was accurately fitted with a single pKa surface protonation model with pK(a) = 5.9 +/- 0.2.     

Capillary zone electrophoresis of cationic and anionic drugs in methanol

The actual mobilities and dissociation constants of acidic and basic pharmaceuticals were determined in methanol. Actual mobilities were derived from the dependence of the effective mobilities of the analytes on the pH of the methanolic background electrolyte solution (pH(MeOH)). The pKa values of the pharmaceuticals in methanol (pK(a,MeOH)) were calculated by non-linear curve fitting to the measured mobility values. It was found that the shift in pKa value (when compounds were transferred from water to methanol) increased with the acidity of the analyte. The average pKa shift for compounds exhibiting acidic properties in water was ca. 5.5 units, and the shift for basic compounds about 2 units. As was shown for a mixture of beta-blockers, the calculated actual mobilities and pKa values can be utilised in the optimisation of pH conditions for separation. The practical value of the method was illustrated by the analysis of urine samples.     

Copper(ii) complexes of a polydentate imidazole-based ligand. pH effect on magnetic coupling and catecholase activity

A potentially dodecadentate N8O4-donor ligand obtained from 2,2'-biimidazole and l-valine and its tetranuclear Cu(ii) complexes in different degrees of protonation were characterized by chemical and spectroscopic methods. The extensive solution studies performed reveal that the rise in pH media leads successively to the formation of imidazolato (pKa(1) and pKa(2) and hydroxido (pKa(3) and pKa(4)) bridges. A frozen solution EPR study shows a decrease in the signal intensity until an EPR silent spectrum is observed, upon increasing the basicity of the solution. The catalytic performance of the oxidation of 3,5-di-tert-butylcatechol to its corresponding quinone was studied using UV-Vis-NIR absorption spectroscopic methods in CH3CN-H2O and in CH3OH-H2O at pH = 7.5, 8.0 and 8.5. A marked increase in activity, consistent with the formation of the hydroxide bridged species, is observed at pH = 8.5 in both solvent mixtures, but the activity is significantly higher in CH3OH-H2O.     

Linear-scaling molecular orbital calculations for the pKa values of ionizable residues in proteins

In this report, we present a computational methodology for the pKa prediction of proteins, based on linear-scaling molecular orbital calculations for their solution-conformations obtained from NMR measurements. The method is used to predict the pKa values of five carboxylic acids (Asp7, Glu10, Glu19, Asp27, and Glu43) in turkey ovomucoid third domain (OMTKY3), and six aspartates residues (Asp 22, Asp 44, Asp 54, Asp 75, Asp 83, and Asp 93) in barnase. For OMTKY3, all the predicted pKa values are within 1 pH units from the available experimental ones, except for the case of Glu 43. For barnase, the root-mean-square deviation from experiment is 1.46 pH units. As a result, the proposed pKa calculation method correctly reproduces the relative order of the pKa values among the carboxylic acids located in different sites of the proteins. The calculated pKa values are decomposed into the contributions of short- and long-range structural difference effects. The results indicate that in both proteins the pKa value of the given carboxylic acid is partially influenced by long-range interactions with distant charged residues, which significantly contribute to determining the relative order of the pKa values. The current methodology based on LSMO provides us useful information about the titration behavior in a protein.     

Surface charge properties of Fe2O3 in aqueous and alcoholic mixed solvents

Iron oxide (Fe2O3) was identified and characterized by surface area, X-ray diffractometry, and FTIR analyses. Surface charge densities, point of zero charge (PZC), and surface ionization constants were determined from the potentiometric titration data in various aqueous and aqueous organic mixed solvents in the temperature range 293-313 K. The surface charge densities were observed to decrease with the increase in temperature and concentration of metal ions in both the aqueous and aqueous organic mixed solvents. The absolute values of the surface charge density were found to change in the order aqueous > aqueous/methanol > aqueous/ethanol. Further, the PZC of the iron oxide was observed to shift to the higher pH values in the order ethanol > methanol > aqueous solution, which indicated a decrease in the acidity of the surface -OH groups. The pKa1 and pKa2 values of iron oxide were also determined and then used for determination of the surface potential (psi0) of the solid in aqueous and aqueous organic mixed solvents. The surface potential-surface charge curves generally supplemented the results derived from psi0-pH curves.     

A perturbed pK(a) at the binding site of the nicotinic acetylcholine receptor: implications for nicotine binding

A series of tethered quaternary ammonium derivatives of Tyr have been incorporated into the binding site of the nicotinic acetylcholine receptor (nAChR) using the in vivo nonsense suppression method, producing constitutively active (self-gating) receptors. We have incorporated primary, secondary, and tertiary amine tethered agonists to give receptors whose constitutive activity can be modulated by pH. Lowering the pH protonates the tethered amine, giving it a positive charge and allowing it to reversibly activate the receptor. Tertiary and secondary tethered amines, TyrO3T and TyrO3S, have been successfully incorporated at alpha149 in the nAChR. Constitutive currents at pH 5.5 are 6 times those at pH 9.0. The pKa of TyrO3T in the binding site appears to be 6 or lower, differing substantially from its pKa in solution ( approximately 9.3). This local pKa perturbation has substantial implications for pharmacological research on the nAChR: of the tertiary agonists considered, noracetylcholine experiences this pKa perturbation, while nicotine does not.     

Evaluation of calibration data in capillary electrophoresis using artificial neural networks to increase precision of analysis

An RP-HPLC study for the pKa determination of a series of basic compounds related to caproctamine, a dibenzylaminediamide reversible inhibitor of acetylcholinesterase, is reported. The 2-substituted analogues, bearing substituents with different electronegativity, were analysed by RP-HPLC by using C18 C4 stationary phases with a mobile phase consisting of mixture of acetonitrile and triethylamine phosphate buffer (pH range comprised between 4 and 10). Typical sigmoidal curves were obtained, showing the dependence of the capacity factors upon pH. In general, the retention of the investigated basic analytes increased with increasing of the pH. The inflection point of the pH sigmoidal dependence was used for the dissociation constant determination at a fixed acetonitrile percentage. When plotting pKa vs. percent of acetonitrile in the mobile phase for two representative compounds, linear regression were obtained: the y intercept gave the aqueous pKa(w). The pKa estimation by HPLC method was found to be useful to underline the difference of benzylamine basicity produced by the ortho aromatic substituents. The variation of pKa values (6.15-7.80) within the series of compounds was correlated with the electronic properties of the ortho-substituents through the Hammett sigma parameter, whereas the ability of substituents to accept H-bond was found to play a role in determining the conformational behavior of the molecules.     

Effects of omega-functional groups on pH-dependent reductive desorption of alkanethiol self-assembled monolayers

Self-assembled monolayers (SAMs) of alkanethiols having various terminal groups on their omega-positions were formed on a Au111 electrode, and their reductive desorption was studied by linear sweep voltammetry, focusing on effects of solution pH on the desorption behavior. The peak potentials (Ep) of cathodic waves representing reductive desorption were found to be reflected by the pKa value of the thiol group and were negatively shifted with an increase in pH of the electrolyte solution. The magnitude of the pH dependency of Ep was greatly influenced by the hydrophobicity of the terminal groups. In the cases of alkanethiol SAMs having pH-sensitive terminal groups such as carboxyl and amino groups, their basicity was estimated from bending points appearing in the pH titration profile of Ep. This method allows direct determination of not only the pKa value of the arrayed groups but also that of the groups dissolved in solution simultaneously. The pKa values of the arrayed carboxyl groups in SAMs were larger by ca. 3 pH units than their original ones, while those for amino groups were smaller by ca. 2 pH units.     

Acid-base titrations of functional groups on the surface of the thermophilic bacterium Anoxybacillus flavithermus: comparing a chemical equilibrium model with ATR-IR spectroscopic data

Acid-base functional groups at the surface of Anoxybacillus flavithermus (AF) were assigned from the modeling of batch titration data of bacterial suspensions and compared with those determined from in situ infrared spectroscopic titration analysis. The computer program FITMOD was used to generate a two-site Donnan model (site 1: pKa = 3.26, wet concn = 2.46 x 10(-4) mol g(-1); site 2: pKa = 6.12, wet concn = 6.55 x 10(-5) mol g(-1)), which was able to describe data for whole exponential phase cells from both batch acid-base titrations at 0.01 M ionic strength and electrophoretic mobility measurements over a range of different pH values and ionic strengths. In agreement with information on the composition of bacterial cell walls and a considerable body of modeling literature, site 1 of the model was assigned to carboxyl groups, and site 2 was assigned to amino groups. pH difference IR spectra acquired by in situ attenuated total reflection infrared (ATR-IR) spectroscopy confirmed the presence of carboxyl groups. The spectra appear to show a carboxyl pKa in the 3.3-4.0 range. Further peaks were assigned to phosphodiester groups, which deprotonated at slightly lower pH. The presence of amino groups could not be confirmed or discounted by IR spectroscopy, but a positively charged group corresponding to site 2 was implicated by electrophoretic mobility data. Carboxyl group speciation over a pH range of 2.3-10.3 at two different ionic strengths was further compared to modeling predictions. While model predictions were strongly influenced by the ionic strength change, pH difference IR data showed no significant change. This meant that modeling predictions agreed reasonably well with the IR data for 0.5 M ionic strength but not for 0.01 M ionic strength.