Thermodynamic, kinetic and pH studies on the reactions of NCS-, N3-, and CH3CO2- with Fusarium galactose oxidase

Thermodynamic and kinetic studies on the X- = NCS-, N3-, and CH3CO2- replacement of H2O/OH- at the CuII exogenous site of the tyrosyl-radical-containing enzyme galactose oxidase (GOaseox) from Fusarium (NRR 2903), have been studied by methods involving UV-vis spectrophotometry (25 degrees C), pH range 5.5-8.7, I = 0.100 M (NaCl). In the case of N3- and CH3CO2- previous X-ray structures have confirmed coordination at the exogenous H2O/OH- site. From the effect of pH on the UV-vis spectrum of GOaseox under buffer-free conditions, acid dissociation constants of 5.7 (pK1a; coordinated H2O) and 7.0 (pK2a; H+Tyr-495) have been determined. At pH 7.0 formation constants K(25 degrees C)/M-1 are NCS- (480), N3- (1.98 x 10(4)), and CH3CO2- (104), and from the variations in K with pH the same two pKa values are seen to apply. No pK1a is observed when X- is coordinated. From equilibration stopped-flow studies rate constants at pH 7.0 for the formation reaction kf(25 degrees C)/M-1 s-1 are NCS- (1.13 x 10(4)) and N3- (5.2 x 10(5)). Both K and kf decrease with increasing pH, consistent with the electrostatic effect of replacing H2O by OH-. In the case of the GOaseox Tyr495Phe variant pK1a is again 5.7, but no pK2a is observed, confirming the latter as acid dissociation of protonated Tyr-495. At pH 7.0, K for the reaction of four-coordinate GOaseox Tyr495Phe with NCS- (1.02 x 10(5) M-1) is more favorable than the value for GOaseox. Effects of H+Tyr-495 deprotonation on K are smaller than those for the H2O/OH- change. The pK1a for GOasesemi is very similar (5.6) to that for GOaseox (both at CuII), but pK2a is 8.0. At pH 7.0 values of K for GOasesemi are NCS- (270 M-1), N3- (4.9 x 10(3)), and CH3CO2- (107).     

Fast high-throughput method for the determination of acidity constants by capillary electrophoresis. II. Acidic internal standards

A fast method for the determination of acidity constants by CZE has been recently developed. This method is based on the use of an internal standard of pK(a) similar to that of the analyte. In this paper we establish the reference pK(a) values of a set of 24 monoprotic neutral acids of varied structure that we propose as internal standards. These compounds cover the most usual working pH range in CZE and facilitate the selection of adequate internal standards for a given determination. The reference pK(a) values of the acids have been established by the own internal standard method, i.e. from the mobility differences between different acids of similar pK(a) in the same pH buffers. The determined pK(a) values have been contrasted to the literature pK(a) values and confirmed by determination of the pK(a) values of some acids of the set by the classical CE method. Some systematic deviations of mobilities have been observed in NaOH buffer in reference to the other used buffers, overcoming the use of NaOH in the classical CE method. However, the deviations affect in a similar degree to the test compounds and internal standards allowing thus, the use of NaOH buffer in the internal standard method. This fact demonstrates the better performance of the internal standard method over the classical method to correct mobility deviations, which together with its fastness makes it an interesting method for the routine determination of accurate pK(a) values of new pharmaceutical drugs and drug precursors.     

Fiber optic pH sensing with long wavelength excitable Schiff bases in the pH range of 7.0-12.0

Most of the fluorescent pH probes work near neutral or acidic regions of the pH scale. In this work, two different fluorescent Schiff bases, chloro phenyl imino propenyl aniline (CPIPA) and nitro phenyl imino propenyl aniline (NPIPA), have been investigated for pH sensing in the alkaline region. Absorption and emission based spectral data, quantum yield, fluorescence lifetime, photostability and acidity constant (pK(a)) of the Schiff bases were determined in conventional solvents and in PVC. The long wavelength excitable immobilized Schiff bases CPIPA (lambda(ex)=556 nm) and NPIPA (lambda(ex)=570 nm) exhibited absorption and emission based optical response to proton in the pH range of 8.0-12.0 and 7.0-12.0, respectively. Response of the CPIPA was fully reversible within the dynamic working range. The response times were between 3-13 min. A relative signal change of 95% and 96% have been achieved for sensor dyes of CPIPA and NPIPA, respectively. The CPIPA displayed better fluorescence quantum yield (varphi(F)=3.7 x 10(-1)) and higher matrix compatibility compared to NPIPA (varphi(F)=1.6 x 10(-1)) in immobilized PVC. The CPIPA and NPIPA exhibited a slight cross sensitivity to the ions of Hg(+) and Fe(3+), respectively.     

Heme-Peptide Models for Hemoproteins. 1. Solution Chemistry of N-Acetylmicroperoxidase-8

An improved method for the preparation of the heme octapeptide acetyl-MP8, obtained by proteolysis of horse heart cytochrome c, is described. AcMP8 obeys Beer's law at pH 7.0 in aqueous solution up to a concentration of 3 x 10(-)(5) M. The self-association constant measured at 25 degrees C (log K(D) = 4.04) is an order of magnitude lower than that for MP8, reflecting the role of the N-acetyl protecting group in abolishing intermolecular coordination. However, AcMP8 does form pi-stacked dimers in aqueous solution with increasing ionic strength. A more weakly packed pi-pi dimer reaches a maximum abundance at approximately 3 M ionic strength, but a more tightly packed dimer is favored at &mgr; > 3 M. An equilibrium model based on charge neutralization by specific binding of Na(+) ions gives a total molecular charge of 3- for AcMP8 at pH 7.0 and a self-association constant log K(D) = 4.20. AcMP8 exhibits six spectroscopically active pH-dependent transitions. The Glu-21 c-terminal carboxylate binds to the heme iron at low pH (pK(a) = 2.1) but is substituted by His-18 (pK(a) = 3.12) as the pH increases. The two heme propanoic acid substituents ionize with pK(a)'s of 4.95 and 6.1. This is followed by ionization of iron-bound water with a pK(a) = 9.59, DeltaH = 48 +/- 1 kJ mol(-)(1), and DeltaS = -22 +/- 3 J K(-)(1) mol(-)(1). The electronic spectra indicate that AcMP8 is predominantly in the S = (5)/(2) state at pH 7.0, while the hydroxo complex at pH 10.5 corresponds to an equilibrium mixture of S = (5)/(2) and S = (1)/(2) states at 25 degrees C. In the final transition, His-18 ionizes to form the S = (1)/(2) histidinate complex with a pK(a) of 12.71. AcMP8 is relatively stable under alkaline conditions, dimerizing slowly at high pH (k = 2.59 +/- 0.14 M(-)(1) s(-)(1)) to form a high-spin &mgr;-oxo-bridged species. The pH-dependent behavior of AcMP8 in the presence of excess 3-cyanopyridine, however, is markedly different. At low pH, AcMP8 simultaneously binds the exogenous ligand and the Glu-21 c-terminal carboxylate with a pK(a) < 2. His-18 replaces the carboxylate ligand at higher pH (pK(a) = 2.60), and both heme propanoic acid groups ionize with a mean pK(a) = 5.10. Unlike AcMP8.OH(-), the axial histidine of the 3-CNPy complex ionizes at near neutral pH (pK(a) = 7.83), prior to being replaced by OH(-) (pK(a) = 10.13). The sixth transition in the AcMP8/3-CNPy system produces the bis(hydroxo) complex (pK(a) > 13).     

Studies on the effects of imidazole on the peroxyoxalate chemiluminescence detection system for high performance liquid chromatography

The catalytic effect of bases (imidazole, pyridine, Tris and triethylamine) on the peroxyoxalate chemiluminescence (PO-CL) reaction for high performance liquid chromatography (HPLC) was investigated. Imidazole increased PO-CL intensity extraordinarily, whereas the other bases (pyridine, Tris and triethylamine) did not. The peak heights of dipyridamole (coronary vasodilator) obtained using the eluents containing buffers were largest at pH 7.0, a few times less at pH 6.0 and pH 5.0, 100 times less at pH 4.0 and a few hundred times less at pH 3.0. The eluents containing buffers at pH 3, 4, 5, 6 or 7 each with imidazole increased the peak heights by a few to ten times as compared with those without imidazole, and those peak heights were within one order of magnitude. On the other hand, the eluent containing buffer at pH 2 did not affect the peak heights with or without imidazole. Bis(4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl) oxalate (TDPO) alone and bis(2,4-dinitrophenyl)oxalate (DNPO) plus TDPO were recommended to be used against eluents containing buffers of pH 5-7 and pH 3-4, respectively. Dipyridamole and benzydamine hydrochloride (anti-inflammatory drug) were separated on the ODS column and detected by the present system. The detection limits of dipyridamole and benzydamine hydrochloride were 40 amol and 270 fmol, respectively.     

Acid-base equilibria in nonpolar media. Absolute pK(a) scale of bases in tetrahydrofuran

The acidity constants (pKa) of 11 bases (amines, anilines, pyridines, pyrrolidines, and iminophosphoranes) have been determined in tetrahydrofuran by potentiometry, complemented by conductometric measurements. The pK(a) values of the studied bases cover a wide absolute pH range of acidity in tetrahydrofuran, from 7.4 to 21.7. From the pK(a) values obtained, a scale of absolute acidity in tetrahydrofuran has been established, which has allowed calculation of the absolute pKa values of 77 bases from literature relative pK(a) data.     

Determination of acid-base dissociation constants of very weak zwitterionic heterocyclic bases by capillary zone electrophoresis

Thermodynamic acid-base dissociation (ionization) constants (pK(a)) of seven zwitterionic heterocyclic bases, first representatives of new heterocyclic family (2,3,5,7,8,9-hexahydro-1H-diimidazo[1,2-c:2',1'-f][1,3,2]diazaphosphinin-4-ium-5-olate 5-oxides), originally designed as chiral Lewis base catalysts for enantioselective reactions, were determined by capillary zone electrophoresis (CZE). The pK(a) values of the above very weak zwitterionic bases were determined from the dependence of their effective electrophoretic mobility on pH in strongly acidic background electrolytes (pH 0.85-2.80). Prior to pK(a) calculation by non-linear regression analysis, the CZE measured effective mobilities were corrected to reference temperature, 25°C, and constant ionic strength, 25 mM. Thermodynamic pK(a) values of the analyzed zwitterionic heterocyclic bases were found to be particularly low, in the range 0.04-0.32. Moreover, from the pH dependence of effective mobility of the bases, some other relevant characteristics, such as actual and absolute ionic mobilities and hydrodynamic radii of the acidic cationic forms of the bases were determined.     

New aspects of buffering with multivalent weak acids in capillary zone electrophoresis: pros and cons of the phosphate buffer

Phosphate buffer is frequently used as background electrolyte in capillary electrophoresis. It can cover a broad range of pH due to the three dissociation constants (pK1 = 2.0, pK2 = 7.2, and pK3 = 11.0) of phosphoric acid and because it is UV-light transparent. This contribution brings a theoretical study of the analytical separation performance (sample window, regions of peak symmetry, regions of fronting and tailing peaks) of phosphate buffer, serving as a model of buffering with multivalent weak acids. The study is based on the use of peak shape diagrams and covers the pH range 2.55-11.43. New important general knowledge has been revealed that single multivalent weak acids mimic the performance of background electrolytes with multiple coanionic species for anionic analyses. It is shown that simple phosphate buffers prepared by mixing phosphoric acid and potassium hydroxide may exhibit up to two regions of symmetry, of fronting as well as of tailing zones, on the mobility scale inside the sample window. Moreover they may exhibit a "schizophrenic" region of enormous electromigration dispersion.     

Effect of ionic strength, pH and polymer concentration on the separation of DNA fragments in the presence of electroosmotic flow

DNA separations in the presence of electroosmotic flow (EOF) using poly(ethylene oxide) (PEO) solutions have been demonstrated. During the separations, PEO entered capillaries filled with Tris-borate (TB) free buffers by EOF and acted as sieving matrices. We have found that ionic strength and pH of polymer and free solutions affect the bulk EOF and resolution differently from that in capillary zone electrophoresis. The EOF coefficient increases with increasing ionic strength of the free TB buffers as a result of decreases in the adsorption of PEO molecules. In contrast, the bulk EOF decreases with increasing the ionic strength of polymer solutions using capillaries filled with high concentrations of free TB buffers. Although resolution values are high due to larger differential migration times between any two DNA fragments in a small bulk EOF using 10 mM TB buffers, use of a capillary filled with at least 100 mM TB free buffers is suggested for high-speed separations. On the side of PEO solutions, 1.5% PEO solutions prepared in 100 to 200 mM TB buffers are more proper in terms of resolution and speed. The separation of DNA markers V and VI was accomplished less than 29 min in 1.5% PEO solutions prepared in 100 mM TB buffers, pH 7.0 at 500 V/cm using a capillary filled with 10 mM free TB buffers, pH 7.0.     

Diprotonation process of meso-tetraphenylporphyrin derivatives designed for photodynamic therapy of cancers: from multivariate curve resolution to predictive QSPR modeling

Tetrapyrrole rings possess four nitrogen atoms, two of which act as Br?ndsted bases in acidic media. The two protonation steps occur on a close pH range, particularly in the case of meso-tetraphenylporphyrin (TPP) derivatives. If the cause of this phenomenon is well known--a protonation-induced distortion of the porphyrin ring--data on stepwise protonation constants and on electronic absorption spectra of monoprotonated TPPs are sparse. A multivariate approach has been systematically applied to a series of glycoconjugated and hydroxylated TPPs, potential anticancer drugs usable in Photodynamic Therapy. The dual purpose was determination of protonation constants and linking substitution with basicity. Hard-modeling version of MCR-ALS (Multivariate Curve Resolution Alternating Least Squares) has given access to spectra and distribution profile of pure components. Spectra of monoprotonated species (H(3)TPP(+)) in solution resemble those of diprotonated species (H(4)TPP(2+)), mainly differing by a slight blue-shift of bands. Overlap of H(3)TPP(+) and H(4)TPP(2+) spectra reinforces the difficulty to evidence an intermediate form only present in low relative abundance. Depending on macrocycle substitution, pK values ranged from 3.5±0.1 to 5.1±0.1 for the first protonation and from 3.2±0.2 to 4.9±0.1 for the second one. Inner nitrogens' basicity is affected by position, number and nature of peripheral substituents depending on their electrodonating character. pK values have been used to establish a predictive Multiple Linear Regression (MLR) model, relying on atom-type electrotopological indices. This model accurately describes our results and should be applied to new TPP derivatives in a drug-design perspective.     

Comparative study of the water oxidizing reactions and the millisecond delayed chlorophyll fluorescence in photosystem II at different pH

Water splitting activity, the multiline EPR signal associated with S(2)-state of the CaMn(4)-cluster and the fast and slow phases of the induction curve of the millisecond delayed chlorophyll fluorescence from photosystem II (PSII) in the pH range of 4.5-8.5 were studied in the thylakoid membranes and purified PSII particles. It has been found that O(2) evolution and the multiline EPR signal were inhibited at acidic (pK approximately 5.3) and alkaline (pK approximately 8.1) pH values, and were maximal at pH 6.0-7.0. Our results indicate that the loss of O(2) evolution and the S(2)-state multiline EPR signal associated with the decrease of the millisecond delayed chlorophyll fluorescence only in alkaline region (pH 7.0-8.5). Possible correlations of the millisecond delayed chlorophyll fluorescence components with the donor side reactions in PSII are discussed.     

Study of overload for basic compounds in reversed-phase high performance liquid chromatography as a function of mobile phase pH

The retention and overloading properties for eight basic solutes and two quaternary ammonium compounds were studied over the pH range 2.7-10.0 using phosphate and carbonate buffers. At low pH, a hybrid inorganic-organic silica-ODS phase (XTerra RP-18, 15 cm x 0.46 cm) showed substantial loss in efficiency when sample masses exceeded about 0.5 microg; these results were similar to those obtained previously on pure silica ODS and wholly polymeric phases, suggesting a common overloading mechanism. At pH 7-8.5, substantial improvements in loading capacity were obtained on XTerra due apparently to the unexpectedly strong influence of small decreases in solute ionisation. Data from the quaternary compounds suggested that silanol ionisation on this phase was still small even at intermediate pH. For many bases, loading capacity continued to improve as the pH was raised to 10, in line with the decrease in the proportion of ionised solute. However, for the highest pK(a) solutes, peak shape worsened at high pH, possibly due to the negative influence of increasing column silanol ionisation.     

Decomposition kinetics of Ni(III)-peptide complexes with histidine and histamine as the third residue

The decomposition kinetics of the Ni(III) complexes of Gly(2)HisGly and Gly(2)Ha are studied from p[H(+)] 3.5 to 10, where His is l-histidine and Ha is histamine. In these redox reactions, at least two Ni(III) complexes are reduced to Ni(II) while oxidizing a single peptide ligand. The rate of Ni(III) loss is first order at low pH, mixed order from pH 7.0 to 8.5, and second order at higher pH. The transition from first- to second-order kinetics is attributed to the formation of an oxo-bridged Ni(III)-peptide dimer. The rates of decay of the Ni(III) complexes are general-base assisted with Br?nsted beta values of 0.62 and 0.59 for Ni(III)Gly(2)HisGly and Ni(III)Gly(2)Ha, respectively. The coordination of Gly(2)HisGly and Gly(2)Ha to Ni(II) are examined by UV-vis and CD spectroscopy. The square planar Ni(II)(H(-2)Gly(2)HisGly)(-) and Ni(II)(H(-2)Gly(2)Ha) complexes lose an additional proton from an imidazole nitrogen at high pH with pK(a) values of 11.74 and 11.54, respectively. The corresponding Ni(III) complexes have axially coordinated water molecules with pK(a) values of 9.37 and 9.44. At higher pH an additional proton is lost from the imidazole nitrogen with a pK(a) value of 10.50 to give Ni(III)(H(-3)Gly(2)Ha)(H(2)O)(OH)(2-).     

Capillary zone electrophoresis of basic analytes in methanol as non-aqueous solvent mobility and ionisation constant

The electrophoretically relevant properties of monoacidic 21 bases (including common drugs) containing aliphatic or aromatic amino groups were determined in methanol as solvent. These properties are the actual mobilities (that of the fully ionised weak bases), and their pKa values. Actual mobilities were measured in acidic methanolic solutions containing perchloric acid. The ionisation constants of the amines were derived from the dependence of the ionic mobilities on the pH of the background electrolyte solution. The pH scale in methanol was established from acids with known conventional pK*a values in this solvent used as buffers, avoiding thus further adjustment with a pH sensitive electrode that might bias the scale. Actual mobilities in methanol were found larger than in water, and do not correlate well with the solvent's viscosity. The pK*a values of the cation acids, HB-, the corresponding form of the base, B, are higher in methanol, whereas a less pronounced shift was found than for neutral acids of type HA. The mean increase (compared to pure aqueous solution) for aliphatic ammonium type analytes is 1.8, for substituted anilinium 1.1, and for aromatic ammonium from pyridinium type 0.5 units. The interpretation of this shift was undertaken with the concept of the medium effect on the particles involved in the acid-base equilibrium: the proton, the molecular base, B, and the cation HB+.     

Peptide separations and dissociation constants in nonaqueous capillary electrophoresis: comparison of methanol and aqueous buffers

Nonaqueous capillary electrophoresis was evaluated for its potential to separate peptides in methanolic background electrolytes in comparison to aqueous-methanol (50% v/v) and water. Isomeric aspartyl dipeptides and Leu- and Met-enkephalin served as model compounds. pK(a) values were determined in the three solvent systems based on the apparent pH scale and in the case of methanol additionally based on the conventional pH scale. Changing from water to methanol led to an increase of the ionization constants describing the dissociation equilibria of the carboxyl group and the amino group, respectively. The pK(a) shift was more pronounced for the carboxylic acid function leading to a compression of the mobility-pH curve. As reported for aqueous buffers, efficient separations of the peptides were achieved in methanolic background electrolytes including the resolution of the diastereomers of the isomeric alpha- and beta-aspartyl dipeptides. In contrast to aqueous buffers, the separation of Leu- and Met-enkephalin could also be obtained in buffers in methanol at high pH.     

Tandem electrostatic effect from the first to the third aglycon in the trimeric RNA owing to the nearest-neighbor interaction

We here show an electrostatic polar-pi interaction from the first to the third aglycon, via the second aglycon, in the ground state in two single stranded trimeric RNAs, 5'-GpA(1)pA(2)-3' (3) and 5'-GpApC-3' (4), as a result of intramolecular nearest neighbor offset-stacking. The experimental evidence in support of this conclusion has been obtained by comparing the pK(a)s of each aglycone in the two trimers with those of guanosine 3'-ethyl phosphate, GpEt (1) and 5'-GpA-3' (2): Thus, the pK(a) of N(1)-H of guanin-9-yl of 5'-GpA(1)pA(2)-3' (3) could be measured by pH titration (pH 7.3-11.6) of its own deltaH8G (pK(a) 9.75 +/- 0.02) as well as from deltaH8A(1) (pK(a) 9.72 +/- 0.02) and deltaH2A(1) (pK(a) 9.83 +/- 0.04) of the neighboring pA(1)p moiety and the deltaH8A(2) (pK(a) 9.83 +/- 0.02) of the terminal pA(2) moiety. Similarly, the pH titration of GpApC (4) shows the pK(a) of N(1)-H of guanin-9-yl from its own deltaH8G (pK(a) 9.88 +/- 0.03) as well as from deltaH8A (pK(a) 9.87 +/- 0.01) of the neighboring pAp moiety, and deltaH5/H6C (pK(a) 9.88 +/- 0.01 and 9.90 +/- 0.01 respectively) of the 3'-terminal cytosin-1-yl. This intramolecular nearest neighbor electrostatic interaction in the single-stranded RNA modulates the pseudoaromaticity of the nearest neighbors by almost total transmission of because they constitute an extended array of offset-stacked coupled aromatic heterocycles within a polyanionic sugar-phosphate backbone at the ground state. The enhanced basicity of Gp residue by ca. 0.6 pK(a) unit in the trimers compared to that of the dimer is a result of the change in the electrostatic microenvironment owing to the nearest neighbors in the former (the nucleobases as well as the phosphates). Thus, the from the 5'-guanylate ion to the 3'-end aglycon via the central adenin-9-yl is 55 to 56 kJ mol(-)(1) in each step through a distance spanning approximately 6.8 A in an unfolded state. As a result, the pK(a) of guanin-9-yl moiety has become 9.25 +/- 0.02 in GpEt (1), 9.17 +/- 0.02 in GpA (2), 9.75 +/- 0.02 in GpApA (3), and 9.88 +/- 0.03 in GpApC (4). This means that guanin-9-yl moiety of trimers 3 and 4 is more basic than in the monomer or the dimer. The net outcome of this electrostatic cross-talk between the two neighboring heterocycles is creation of new hybrid aglycones in an oligo or polynucleotide, whose physicochemical property and the pseudoaromatic character are completely dependent both upon the nearest neighbors, and whether they are stacked or unstacked. Thus, this tunable physicochemical property of an aglycon (an array of the extended genetic code) may have considerable implication in our understanding of the specific ligand binding ability of an aptamer, the pK(a) and the hydrogen bonding ability of nucleic acids in a microenvironment, or in the triplet usage by the anticodon-codon interaction in the protein biosynthesis in the ribosome.     

Extension of the self-consistent spectrophotometric basicity scale in acetonitrile to a full span of 28 pKa units: unification of different basicity scales

The earlier compiled self-consistent spectrophotometric basicity scale in acetonitrile (AN) was expanded to range from 3.8 to 32.0 pK(a) units, that is 28 orders of magnitude. Altogether 54 new relative basicity measurements (DeltapK(a) measurements) were carried out and 37 new compounds were introduced to the scale (it now includes altogether 89 bases). The relative basicity of any two bases in the scale can be obtained by combining at least two independent sets of measurements. Multiple overlapping measurements make the results more reliable. The overall consistency (as defined earlier) of the measurements is s = 0.03 pK(a) units. Thorough analysis of all of our experimental data (DeltapK(a) values of this and earlier works) and experimental pK(a) data in AN available in the literature (works from the groups of Coetzee and Padmanabhan, Kolthoff and Chantooni, Jr., the Schwesinger group, Bren' et al. and some others, altogether 19 papers) was carried out. On the basis of this analysis the anchor point of the scale-pyridine-was shifted upward by 0.20 pK(a) units thereby also revising the absolute pK(a) values of all the bases on the scale. This way very good agreement between our relative data and the absolute pK(a) values of the abovementioned authors was obtained. The revised basicity scale was interconnected with the earlier published self-consistent acidity scale by DeltapK(a) measurements between acids and bases. The rms deviation between the directly measured DeltapK(a) values and the absolute pK(a) values of the compounds was 0.10 pK(a) units.     

Effect of substitution of proline-77 to aspartate on the light-driven proton release of bacteriorhodopsin

Wild-type bacteriorhodopsin (BR) and another retinal protein archaerhodopsin 4 (AR4) are both light-driven proton pumps, but exhibit opposite temporal orders of proton release and uptake upon a flash illumination at neutral pH due to a higher pK(a) of proton release complex (PRC) in AR4. Since the 77th residue in the extracellular side is proline (P) in BR, but aspartic acid (D) in AR4, we have mutated P77 in BR by D in this study. The new point mutation was found to affect the kinetics of proton release and the pH dependence significantly. Upon a flash excitation, three components "fast proton release,"proton uptake" and "slow proton release" were observed at neutral pH in P77D. The pK(a) of PRC in the M intermediate was increased from 5.6 in the wild-type to 7.0, and became closer to that in AR4, which is 8.4. The coupling strength between D85 and PRC were also weakened, as expected. These data indicate that the 77th residue in AR4 greatly account for the difference between the two proton pumps.     

Interaction of Chlorpromazine and Trifluoperazine with Anionic Sodium Dodecyl Sulfate (SDS) Micelles: Electronic Absorption and Fluorescence Studies

The characteristics of binding of two phenothiazine antipsychothic drugs, chlorpromazine (CPZ) and trifluoperazine (TFP), to anionic sodium dodecyl sulfate (SDS) monomers and/or micelles were investigated using electronic absorption and fluorescence spectroscopies. Binding constants K(b) and pK(a) values for the drugs in SDS micelles were estimated using the red shifts of the maximum absorption and changes in absorption upon alkalization or in the presence of surfactant. The pK(a) shift of CPZ due to its interaction with SDS micelles is about 0.7 unit to higher values, as compared to the reported value of pK(a) obtained in buffer around 9.3. For TFP the pK(a) shift is 0.4 unit to higher values compared to that in buffer, reported as 4.0. The electronic absorption spectroscopic data suggest a biphasic interaction as a function of detergent concentration which is quite dependent of the protonation states of the drugs. In the case of TFP a very strong binding takes place when the drug is fully protonated (pH 2.0) and a distinct binding takes place at stoichiometric (low) surfactant concentrations (interaction via surfactant monomers) and at higher concentrations (in the presence of micelles). Static fluorescence probe analysis using pyrene was used to study the nature of the phenothiazine-surfactant premicellar and self-aggregates. The I(3)/I(1) and I(475)/I(1) ratios associated to pyrene fluorescence vibronic bands and excimer intensities ratios, respectively, were monitored for several ratios [SDS]/[drug] and significant changes, dependent of the drug presence and its protonation state, have been observed revealing a hydrophobic microenvironment provided by TFP-SDS aggregates in comparison with CPZ both at pH 7.0 and 4.0. Static anisotropy was also used to monitor the changes of the self-aggregates and micellar packing in the presence of the phenothiazine drugs. In aqueous solutions the anisotropy of the fluorescent probe dipyridamole (DIP) is quite low, being around 0.005 at pH 7.0 and 0.025 at pH 4.0, and the addition of detergent leads to an increase in the values of anisotropy to 0.030 at pH 7.0 and 0.070 at pH 4.0. In the presence of the phenothiazine drugs, and in the premicellar detergent concentration range, the anisotropy of DIP increases to 0.134 and 0.111 (dependent on drug concentration) for CPZ and TFP, respectively, at pH 4.0. These results suggest that the presence of both phenotiazine drugs makes the premicellar aggregates more rigid by decreasing the probe mobility, and are consistent with a more polar localization of the CPZ in the micelles as compared with TFP. At pH 7.0 the anisotropy changes are smaller, suggesting a slight decrease in CMC induced by the phenothiazines. Copyright 2000 Academic Press.     

Copper trafficking mechanism of CXXC-containing domains: insight from the pH-dependence of their Cu(I) affinities

Copper is trafficked to cellular destinations by homeostatic proteins that also prevent adverse reactivity of the metal. The copper metallochaperone HAH1 (human Atx1) binds Cu(I) via a CXXC motif on loop1/α1 of a βαββαβ ferredoxin-like structure. A similar fold constitutes each of the six metal-binding domains (MBDs) of the two P-type ATPases (Menkes and Wilson disease proteins), the destination for copper bound to HAH1. In this work we have investigated the influence of pH on copper trafficking between HAH1 and the first MBD of the Menkes protein (MNK1). Cu(I) affinities of 5.6 × 10(17) and 3.6 × 10(17) M(-1) have been determined at pH 7.0 for HAH1 and MNK1, respectively, from competition titrations with the chromophoric Cu(I) ligand bathocuproine disulfonate. The mutation of Lys60 on loop5 of HAH1 to Ala (the corresponding residue is Phe67 in MNK1) results in a 3-fold lowering of the affinity for Cu(I) at pH 7.0. The Cu(I) affinity of WT HAH1 exhibits a different pH-dependence compared to MNK1 and Lys60Ala HAH1. This arises because the pK(a) of the second Cys ligand in the CXXC motif of HAH1 is 1.5 pH units lower due to stabilization of the thiolate via a hydrogen-bonding interaction with the side chain of Lys60. The thermodynamic gradient for Cu(I) transfer between HAH1 and MNK1 depends on pH. The decrease in the pK(a) of the Cys ligand in HAH1 can also influence the kinetics of Cu(I) transfer.