H2O Buffer Research Articles

Application of dopamine as an electroactive ligand for the determination of aluminum in biological fluids.

Dopamine (3,4-dihydroxyphenylethylamine, DA) is applied as an electroactive chelant for indirect determination of aluminum (Al) in biological fluids. It is observed that the decrease of the differential pulse voltammetric (DPV) anodic peak current of DA is linear with the increase of Al concentration. Under optimum experimental conditions (pH 8.6, 2.0 x 10(-4) M DA, and 0.03 M NH4Ac-NH3 x H2O buffer solution), two linear ranges, 5.0 x 10(-8) - 4.0 x 10(-7) M and 4.0 x 10(-7) - 7.2 x 10(-6) M Al(III), are obtained. The detection limit of Al is 1.9 x 10(-8) M and the relative standard deviation for 4 x 10(-6) M Al(III) is 3.1% (N = 8). Many biologically active foreign species have been selected for interference. Excellent recoveries and accuracy have been obtained in the measurements of Al in biological samples such as synthetic renal dialysate, Ringer's solution, human whole blood, cerebrospinal fluid of demented patient, and urine of diabetic patient. The methodological principle that Al complexes with DA on the electroactive position result in the depression of electrochemical activities of DA has been verified by comparing both the electrochemical behaviors and the spectroscopic responses like UV-vis and Raman of DA in the presence and in the absence of Al.

Standardization of pH measurements based on the ion interaction approach

The Pitzer method was used to calculate the pH values on the conventional and “true” scales for the TRIS—TRIS·HCl−NaCl−H2O buffer system in the 0–40 °C temperature region and 0–4 NaCl molality interval. This buffer can be used as a standard for pH measurements in a wide range of ionic strengths. The conventional scale is used in cells without a salt bridge. The “true” scale is recommended for pH measurements using cells with a salt bridge. At the same concentrations of the buffer solution, the “true” scale is essentially transformed into the scale of the National Bureau of Standards (NBS) of the USA.

Non-ideal properties of the TRIS—TRIS·HCl−NaCl−H2O buffer system in the 0–40 °C temperature interval. Application of the pitzer equations

The buffer solution TRIS—TRIS·HCl−NaCl−H2O was studied in the 0–40 °C temperature region and ionic strength interval of (0.1–4)m (m is molality) by the e.m.f. method using two types of cells without liquid junction composed of platinum-hydrogen, silverchloride, and sodium-glass electrodes. For temperatures of 5 and 15 °C and the (1–4)m concentration region, the osmotic coefficients of the TRIS·HCl−H2O solutions were measured by the isopiestic method. The results were processed in the framework of the Pitzer method, and the parameters of interaction of the components of the buffer system were calculated. The associative character of the interactions in the TRIS·HCl−H2O solution was shown.

Differential pulse voltammetric indirect determination of aluminium in drinking waters, blood, urine, hair, and medicament samples using L-dopa under alkaline conditions.

The differential pulse voltammetric (DPV) indirect determination of aluminium using L-dopa under alkaline conditions on a glassy carbon working electrode was studied. The proposed method relies on the linear decrease of the DPV anodic peak current of L-dopa with increase in the concentration of aluminium added. Under the optimum experimental conditions (pH 8.5, 0.08 M NH4Cl-NH3.H2O buffer solution, and 4 x 10(-4) M L-dopa), the linear range is 2-18 x 10(-7) M AlIII. The detection limit is 7.6 x 10(-8) M and the relative standard deviation for 8 x 10(-7) M AlIII is 3.5% (n = 8). A number of foreign species were examined as potential interferents. The method was applied to the determination of aluminium in drinking waters, synthetic renal dialysate, sodium chloride injection, sucrafate, hydrothorax, blood, urine and hair samples. The physiological significance is discussed.

1H NMR studies on the CuA center of nitrous oxide reductase from Pseudomonas stutzeri.

1H NMR spectra of the CuA center of N2OR from Pseudomonas stutzeri, and a mutant enzyme that contains only CuA, were recorded in both H2O- and D2O-buffered solution at pH 7.5. Several sharp, well-resolved hyperfine-shifted 1H NMR signals were observed in the 60 to -10 ppm chemical shift range. Comparison of the native and mutant N2OR spectra recorded in H2O-buffered solutions indicated that several additional signals are present in the native protein spectrum. These signals are attributed to a dinuclear copperII center. At least two of the observed hyperfine-shifted signals associated with the dinuclear center, those at 23.0 and 13.2 ppm, are lost upon replacement of H2O buffer with D2O buffer. These data indicate that at least two histidine residues are ligands of a dinuclear CuII center. Comparison of the mutant N2OR 1H NMR spectra recorded in H2O and D2O indicates that three signals, c (27.5 ppm), e (23.6 ppm), and i (12.4 ppm), are solvent exchangeable. The two most strongly downfield-shifted signals (c and e) are assigned to the two N epsilon 2H (N-H) protons of the coordinated histidine residues, while the remaining exchangeable signal is assigned to a backbone N-H proton in close proximity to the CuA cluster. Signal e was found to decrease in intensity as the temperature was increased, indicating that proton e resides on a more solvent-exposed histidine residue. One-dimensional nOe studies at pH 7.5 allowed the histidine ring protons to be definitively assigned, while the remaining signals were assigned by comparison to previously reported spectra from CuA centers. The temperature dependence of the observed hyperfine-shifted 1H NMR signals of mutant N2OR were recorded over the temperature range of 276-315 K. Both Curie and anti-Curie temperature dependencies are observed for sets of hyperfine-shifted protons. Signals a and h (cysteine protons) follow anti-Curie behavior (contact shift increases with increasing temperatures), while signals b-g, i, and j (histidine protons) follow Curie behavior (contact shift decreases with increasing temperatures). Fits of the temperature dependence of the observed hyperfine-shifted signals provided the energy separation (Delta EL) between the ground (2B3u) and excited (2B2u) states. The temperature data obtained for all of the observed hyperfine-shifted histidine ligand protons provided a Delta EL value of 62 +/- 35 cm-1. The temperature dependence of the observed cysteine C beta H and C alpha H protons (a and h) were fit in a separate experiment providing a Delta EL value of 585 +/- 125 cm-1. The differences between the Delta EL values determined by 1H NMR spectroscopy and those determined by EPR or MCD likely arise from coupling between relatively low-frequency vibrational states and the ground and excited electronic states.

Stabilization of tubulin by deuterium oxide.

Tubulin is an unstable protein when stored in solution and loses its ability to form microtubules rapidly. We have found that D2O stabilizes the protein against inactivation at both 4 and 37 degrees C. In H2O-based buffer, tubulin was completely inactivated after 40 h at 4 degrees C, but in buffer prepared in D2O, no activity was lost after 54 h. Tubulin was completely inactivated at 37 degrees C in 8 h in H2O buffer, but only 20% of the activity was lost in D2O buffer. Tubulin also lost its colchicine binding activity at a slower rate in D2O. The deuterated solvent retarded an aggregation process that occurs during incubation at both temperatures. Inactivation in H2O buffer was partially reversed by transferring the protein to D2O buffer; however, aggregation was not reversed. The level of binding of BisANS, a probe of exposed hydrophobic sites in proteins, increases during the inactivation of tubulin. In D2O, the rate of this increase is slowed somewhat. We propose that D2O has its stabilizing effect on a conformational step or steps that involve the disruption of hydrophobic forces. The conformational change is followed by an aggregation process that cannot be reversed by D2O. As reported previously [Ito, T., and Sato, H. (1984) Biochim. Biophys. Acta 800, 21-27], we found that D2O stimulates the formation of microtubules from tubulin. We also observed that the products of assembly in D2O/8% DMSO consisted of a high percentage of ribbon structures and incompletely folded microtubules. When these polymers were disassembled and reassembled in H2O/8% DMSO, the products were microtubules. We suggest that the combination of D2O and DMSO, both stimulators of tubulin assembly, leads to the rapid production of nuclei that lead to the formation of ribbon structures rather than microtubules.

Topology of sarcoplasmic reticulum Ca2+-ATPase: an infrared study of thermal denaturation and limited proteolysis.

Sarcoplasmic reticulum Ca2+-ATPase structure and organization in the membrane has been studied by infrared spectroscopy by decomposition of the amide I band. Besides the component bands assignable to secondary structure elements such as alpha-helix, beta-sheet, etc...., two unusual bands, one at 1,645 cm(-1) in H2O buffer and the other at 1,625 cm(-1) in D2O buffer are present. By perturbing the protein using temperature and limited proteolysis, the band at 1,645 cm(-1) is tentatively assigned to alpha-helical segments located in the cytoplasmic domain and coupled to beta-sheet structure, whereas the band at 1,625 cm(-1) arises probably from monomer-monomer contacts in the native oligomeric protein. The secondary structure obtained is 33% alpha-helical segments in the transmembrane plus stalk domain; 20% alpha-helix and 22% beta-sheet in the cytoplasmic domain plus 19% turns and 6% unordered structure. Thermal unfolding of Ca2+-ATPase is a complex process that cannot be described as a two-state denaturation. The results obtained are compatible with the idea that the protein is an oligomer at room temperature. The loss of the 1,625 cm(-1) band upon heating would be consistent with a disruption of the oligomers in a process that later gives rise to aggregates (appearance of the 1,618 cm(-1) band). This picture would also be compatible with early results suggesting that processes governing Ca2+ accumulation and ATPase activity are uncoupled at temperatures above 37 degrees C, so that while ATPase activity proceeds at high rates, Ca2+ accumulation is inhibited.

Solution Conformation of the (-)-trans-anti-5-Methylchrysene-dG Adduct opposite dC in a DNA Duplex: DNA Bending Associated with Wedging of the Methyl Group of 5-Methylchrysene to the 3'-Side of the Modification Site

This paper reports on NMR-molecular mechanics structural studies of the (-)-trans-anti-[MC]dG adduct positioned opposite dC in the sequence context of the d(C1-C2-A3-T4-C5-[MC]G6-C7-T8-A9-C10-C11).d(G12-G13-T14++ +-A15-G16-C17-G18- A19-T20-G21-G22) duplex [designated (-)-trans-anti-[MC]dG.dC 11-mer duplex]. This adduct is derived from the trans addition at C4 of (-)-anti-1(S),2(R)-dihydroxy-3(R),4(S)-epoxy-1,2,3,4-tetrahydro-5-met hylchrysen e [(-)-anti-5-MeCDE] to the N2 position of dG6 in this duplex sequence. The 5-methyl group is located adjacent to the MC(C4) binding site, with these groups juxtaposed in a sterically crowded bay region in the adduct duplex. The 5-methylchrysenyl and the nucleic acid exchangeable and nonexchangeable protons were assigned following analysis of two-dimensional NMR data sets in H2O and D2O buffer solution. The solution structure of the (-)-trans-anti-[MC]dG.dC 11-mer duplex has been determined by incorporating DNA-DNA and carcinogen-DNA proton-proton distances defined by lower and upper bounds deduced from NOESY data sets as restraints in molecular mechanics computations in torsion angle space. The results establish that the [MC]dG6.dC17 base pair and flanking dC5.dG18 and dC7.dG16 base pairs retain Watson-Crick alignments upon adduct formation. The aromatic chrysenyl ring is positioned in the minor groove of a right-handed B-DNA helix and stacks predominantly over the sugar of the dC17 residue across from it on the unmodified complementary strand. The chrysenyl ring points toward the 3'-end of the modified strand with its 5-methyl group inserting between the modified [MC]dG6.dC17 and dC7.dG16 base pairs. The adduct duplex bends by approximately 47 degrees as a result of the wedged insertion of the 5-methyl group from the minor groove face of the duplex. The solution structure of the (-)-trans-anti-[MC] dG.dC 11-mer duplex is compared with that of the corresponding (-)-trans-anti-[BP]dG.dC 11-mer [De los Santos et al. (1992) Biochemistry 31, 5245-5252] in which the [BP]dG adduct is derived from the binding of (-)-anti-BPDE [7(S),8(R)-dihydroxy-9(R),10(S)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene] to the N2 position in the same DNA sequence context. Although the solution structures of the (-)-trans-anti-stereoisomers of 5-methylchrysenyl-dG and benzo[a]pyrenyl-dG adducts opposite dC exhibit many features in common with each other, the [MC]dG adduct which contains a bay region methyl group bends the DNA helix to a greater extent than in the corresponding [BP]dG adduct, which lacks a bay region methyl group.(ABSTRACT TRUNCATED AT 400 WORDS)

Hydration and order in lipid bilayers.

The relationship between membrane lipid bilayer hydration and acyl chain order was investigated using time-resolved fluorescence spectroscopy. The degree of hydration in the head group region was assessed from fluorescence lifetime data along with fluorescence intensity measurements in D2O, relative to H2O buffer, using N-(5-dimethylaminonaphthalene-1-sulfonyl)dipalmitoylphosphatidylethan ola mine (dansyl-PE). The degree of hydration in the acyl chain region was estimated from its effect on the fluorescence lifetime of 1-palmitoyl-2-[[2-[4-(6-phenyl-trans-1,3,5-hexatrienyl)phenyl]ethyl] carbonyl]-3-sn-phosphatidylcholine (DPH-PC), and acyl chain order was determined from time-resolved anisotropy measurements of the DPH-PC. Comparisons of sn-2 unsaturation with sn-1,2 diunsaturation in phosphatidylcholine (PC) bilayers with the same number of double bonds/PC revealed a marked difference in interchain hydration and acyl chain order but little difference in terms of head group hydration. For diunsaturated dioleoyl-PC (DOPC) bilayers with two double bonds/PC, the DPH-PC fluorescence lifetime data indicated a greater level of interchain hydration than 1-palmitoyl-2-docosahexaenoyl-PC (PDPC) with six double bonds/sn-2 chain. By contrast, the head group hydration for DOPC was markedly less than for PDPC. A similar lack of correlation of effects on the two regions of the bilayer was found with cholesterol, it having opposite effects on interchain and head group hydration. When DPH-PC fluorescence lifetime data for bilayers composed of a range of different lipids was plotted as a function of acyl chain order, a strong correlation of interchain hydration with acyl chain order was revealed that was independent of lipid composition.(ABSTRACT TRUNCATED AT 250 WORDS)

Solution conformation of the (-)-trans-anti-benzo[c]phenanthrene-dA ([BPh]dA) adduct opposite dT in a DNA duplex: intercalation of the covalently attached benzo[c]phenanthrenyl ring to the 3'-side of the adduct site and comparison with the (+)-trans-anti-[BPh]dA opposite dT stereoisomer.

This paper reports on NMR-molecular mechanics structural studies of the (-)- trans-anti-benzo[c]phenanthrene-dA adduct positioned opposite dT in the sequence context of the d(C1-T2-C3-T4-C5-[BPh]A6-C7-T8-T9-C10-C11).d(G12- G13-A14-A15-G16-T17-G18-A19-G20-A21- G22) duplex (designated as the (-)-trans-anti-[BPh]dA.dT 11-mer duplex). This adduct is derived from the covalent binding of (-)-1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydro-benzo[c]phenanthrene [(-)-anti-BPhDE] to N6 of dA6 in this duplex sequence. The benzo[c]phenanthrenyl and nucleic acid exchangeable and nonexchangeable protons were assigned in the predominant conformation following analysis of two-dimensional NMR data sets in H2O and D2O buffer solution. The solution structure of the (-)-trans-anti-[BPh]dA.dT 11-mer duplex has been determined by incorporating intramolecular and carcinogen-DNA proton-proton distances defined by lower and upper bounds deduced from NOESY data sets as restraints in molecular mechanics computations in torsion angle space. The results show that the [BPh]dA6.dT17 base pair propeller twists and buckles slightly to permit the covalently attached benzo[c]phenanthrenyl ring to intercalate between the [BPh]dA6.dT17 and dC7.dG16 base pairs to the 3'-side of the [BPh]dA6 lesion site without disrupting the Watson-Crick hydrogen bond alignments in the modified duplex. The strain in the highly sterically hindered fjord region of the benzo[c]phenanthrenyl moiety is relieved by the propeller-like nonplanar geometry of the aromatic phenanthrenyl ring system, which stacks predominantly with the dG16 and dT17 bases on the unmodified strand. The benzylic ring adopts a distorted half-chair form, in which the H1 and H2 protons are pseudo-diequatorial and the H3 and H4 protons are pseudodiaxial. The current observation that the (-)-trans-anti-[BPh]dA positioned opposite dT intercalates to the 3'-side of the intact modified base pair contrasts with our previous demonstration that the stereoisomeric (+)-trans-anti-[BPh]dA adduct positioned opposite dT intercalates to the 5'-side of the intact modified base pair [Cosman, M., et al. (1993b) Biochemistry 32, 12488-12497]. These stereochemically induced structural differences between isomeric [BPh]dA lesions derived from the binding of chiral (+)- and (-)-anti-BPhDE enantiomers may in turn profoundly influence the interactions of the carcinogen-modified DNA with repair and replication enzymes in the cell.

Homosynaptic facilitation of transmitter release in crayfish is not affected by mobile calcium chelators: implications for the residual ionized calcium hypothesis from electrophysiological and computational analyses

1. Evoked neurotransmitter release at the crayfish neuromuscular junction was measured in the presence of the cell-permeant calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetotoxymethyl (BAPTA-AM). Excitatory post-synaptic potentials were greatly diminished after application of the intracellular chelator, an effect resulting from attenuation of the rise in the concentration of cytoplasmic Ca2+ ([Ca]i) that is necessary for neurotransmission. However, short-term homosynaptic facilitation of release, the magnitude and time course of which is thought to depend on the accumulation and removal of residual Ca ions (Ca2+), was not affected. Application of the cell-permeant form of ethylene glycol-bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) gave similar results. 2. To interpret these results we developed a reaction-diffusion model in 3D rectangular coordinates for Ca2+ diffusion in the presence of mobile and immobile buffers. Solutions of the model in response to influx of Ca2+ through one or six channels for different diffusion coefficients and no nondiffusable buffer, predict that 1) the time course of residual Ca2+ is very brief, 2) an unrealistically low Ca2+ diffusion coefficient is required for residual calcium, 3) the spatially distributed Ca2+ signal is attenuated by intracellular BAPTA, 4) the rate at which free Ca2+ returns to resting levels, after entry (residual Ca2+) is faster with more mobile buffer, and 5) when pulse trains of Ca2+ channel current are used as input, computed facilitation is comparable to experimental measurements without buffer, but is abolished in the presence of exogenous buffer. 3. When the diffusion coefficient of Ca2+ in water is used, there is no residual Ca2+; however, when 0.1-1.6 mM nondiffusable buffer is present with a fast binding coefficient comparable to BAPTA, there is a very small residual Ca2+ due to the unbinding from the fixed binding sites. The nondiffusable buffer is saturated next to a Ca2+ channel. For this case of the diffusion coefficient of calcium in H2O and nondiffusable buffer, when a moderate amount of diffusable buffer is added to the system containing nondiffusable buffer, the very small residual Ca2+ is substantially reduced. This is because the product of diffusable buffer and Ca2+ is carried away as diffusable product, in contrast to the nondiffusable product releasing Ca2+, after Ca2+ entry ceases. 4. The model predicts that mobile calcium buffers with appropriate physical properties will attenuate facilitation and hasten its decay by removing residual calcium.(ABSTRACT TRUNCATED AT 400 WORDS)

Secondary structure and temperature behaviour of acetylcholinesterase

The secondary structure of the acetylcholinesterase and its temperature behaviour have been investigated using Fourier-transform infrared (FTIR) spectroscopy. The data are compared to the structure obtained by X-ray analysis of the crystalline enzyme. The secondary structure was determined using the spectral features observed in the amide-I band (H2O buffer) and amide-I' band (D2O buffer) at 1600-1700 cm-1, taking advantage of resolution-enhancement techniques along with least-squares band-fitting procedures. The relative amounts of different secondary-structure elements, 34-36% for alpha-helices, 19-25% for beta-sheets, 15-16% for turns and 13-17% for irregular structures, were estimated. These data, obtained with the enzyme in solution, correlate well with X-ray data of the crystalline protein [Sussman, J. L., Hard, M., Frolow, F., Oefner, C., Goldman, A., Toker, L. & Silman, I. (1991) Science 253, 872-879]. These results are also in good agreement with those obtained by computing the psi and phi angles of the peptide backbone using the Kabsch and Sanders method [Kabsch, W. & Sanders, C. (1983) Biopolymers 22, 2577-2637]. In conjunction with the X-ray data, two bands in the FTIR spectra were assigned to different populations of long and short alpha-helices. Until now this phenomenon has only been described by theoretical calculations [Nevskaya, N. A. & Chirgadze, Yu. N. (1976) Biopolymers 15, 637-648]. The relationship between the thermally induced loss of enzyme activity and secondary-structure changes has also been investigated. The decrease in enzyme activity to zero at 30-40 degrees C was accompanied only by minor changes in the secondary structure. At 55-60 degrees C, denaturation of AChE occurs. In this temperature range, all bands assigned to the various secondary-structure elements abruptly disappear in a co-operative and irreversible manner, whereas the beta-aggregation bands (at 1622 cm-1 and the corresponding high-frequency band) increase in intensity at the same rate.

A neutron solution scattering study of the structure of annexin-V and its binding to lipid vesicle

Low-angle neutron solution scattering has been used to study the structure of annexin-V and its interaction with small single-bilayer vesicles consisting of phosphatidylserine and phosphatidylcholine at a 33:66 (mol:mol) ratio. There was no evidence for a change in the state of aggregation of annexin-V, which remained as a monomer in the presence of 3 mM-free calcium. The only difference between presence and absence of free calcium was the increase of the radius of gyration, from 19(+/- 0.4) A to 22(+/- 0.4) A in 2H2O buffer and from 19.7(+/- 1.2) A to 22.2(+/- 1.2) A in H2O buffer. The relative molecular weight, outer radius and average surface area per lipid of vesicles alone were respectively 2.5(+/- 0.5) x 10(6), 127 A and 90(+/- 19) A2. These parameters were not modified in the presence of free calcium, which testified to the absence of vesicle coalescence. The calcium-dependent binding of annexin-V was essentially interfacial and therefore did not alter significantly the structural characteristics of the vesicles. At saturation, 80(+/- 10) annexin-V molecules were bound per vesicle, the available area per molecule being 2500(+/- 300) A2 thus covering approximately 28 lipid head groups. The protein shell was approximately 35 A thick. The apparent dissociation constant was probably less than 1 nM. These data contribute to a more accurate definition of annexin-V as a possible probe of those cytodynamic events involving exposure of sequestered membrane aminophospholipids.

Synthesis of Chiral 12‐Phenyl(2H)dodecanoic Acids: Useful metabolic probes for the biosynthesis of 1‐alkenes from fatty acids

AbstractThe synthesis of chiral 12‐phenyi(2H)dodecanoic acids as metabolic probes for the evaluation of the stereo‐chemical course of the biosynthesis of 1‐alkerses from fatty acids in plants and insects is described. The diastereoisomeric (2R, 3R)‐ or (2S, 3S)‐12‐phenyl(2,3−2H2)dodecanoic acids 11 are obtained in high chemical and optical yield (>97% e.e.) from the readily available (E)‐12‐phenyl(2,3‐2H2)dodec‐2‐enoic acid (10) or (E)‐12‐phenyldodec‐2‐enoic acid (10a) by microbial reduction with wet packed cells of Clostridium tyrobutyricum in either 2H2O or H2O buffer. (2R)‐ and (2S)‐12‐phenyl(2−2H)dodecanoic acids 9 (>97% e.e.) are accessible from the allylic alcohol 6 via Sharpless epoxidation with (+)‐L‐ or (−)‐D‐diethyl tartrate, Synthetic routes to the (E)‐ and (Z)‐11‐phenyl(1−2H) undec‐1‐enes 16 and 16a as reference compounds are also included.

Simultaneous optical measurement of osmotic and diffusional water permeability in cells and liposomes

A quantitative description of transmembrane water transport requires specification of osmotic (Pf) and diffusional (Pd) water permeability coefficients. Methodology has been developed to measure Pf and Pd simultaneously on the basis of the sensitivity and rapid response of the fluorophore aminonaphthalenetrisulfonic acid (ANTS) to solution H2O/D2O content. Cells loaded with ANTS in an H2O buffer were subjected to an inward osmotic gradient with a D2O buffer in a stopped-flow apparatus. The time courses of cell volume (giving Pf) and H2O/D2O content (giving Pd) were recorded with dual photomultiplier detection of scattered light intensity and ANTS fluorescence, respectively. The method was validated by using sealed red cell ghosts and artificial liposomes reconstituted with the pore-forming agent gramicidin D. At 25 degrees C, red cell ghost Pf was 0.021 cm/s with Pd 0.005 cm/s (H2O/D2O exchange time 7.9 ms). Pf and Pd were inhibited by 90% and 45% upon addition of 0.5 mM HgCl2. The activation energy for Pd increased from 5.1 kcal/mol to 10 kcal/mol with addition of HgCl2 (18-35 degrees C). In 90% phosphatidylcholine (PC)/10% cholesterol liposomes prepared by bath sonication and exclusion chromatography, Pf and Pd were 5.1 X 10(-4) and 6.3 X 10(-4) cm/s, respectively (23 degrees C). Addition of gramicidin D (0.1 micrograms/mg of PC) resulted in a further increment in Pf and Pd of 7 X 10(-4) and 3 X 10(-4) cm/s, respectively. These results validate the new methodology and demonstrate its utility for rapid determination of Pf/Pd in biological membranes and in liposomes reconstituted with water channels.

Direct fluorescence measurement of diffusional water permeability in the vasopressin-sensitive kidney collecting tubule

A fluorescence method has been developed for accurate and instantaneous measurement of transepithelial diffusional water permeability (Pd) in perfused kidney tubules based on the sensitivity of the fluorophore aminonapthelane trisulfonic acid (ANTS) to solution H2O/D2O content. The fluorescence of ANTS was 3.2-fold lower in an H2O buffer than in a D2O buffer. The response of ANTS fluorescence to a change in solution H2O/D2O content occurred in less than 1 ms and was due to a collisional quenching mechanism. Isolated cortical (CCT) and outer medullary (OMCT) collecting tubules from rabbit were perfused with an isosmotic D2O buffer at specified lumen flow rates (2-100 nl/min); tubules were bathed in isosmotic H2O or D2O buffers in which vasopressin (VP) could be added rapidly. Lumen fluorescence was monitored by quantitative epifluorescence microscopy at 380 +/- 5 nm excitation and greater than 530 emission wavelengths. Pd was determined from tubule geometry, lumen flow, ANTS fluorescence, and ANTS fluorescence vs. H2O/D2O calibration relation. The instrument response time for a change in bath H2O/D2O content was less than 4 s. At 37 degrees C, Pd values (mean +/- SE in cm/s x 10(4] were 6.4 +/- 1.0 (-VP, n = 9) and 14.3 +/- 1.1 (+250 microU/ml bath VP, n = 9) in the CCT, and 5.8 +/- 1.0 (-VP, n = 6) and 15.3 +/- 2.0 (+VP, n = 6) in the OMCT; at 23 degrees C, Pd was 5.1 +/- 0.6 (-VP, n = 4) and 7.8 +/- 0.6 (+VP, n = 4) in the CCT. In response to rapid addition of 250 micro U/ml vasopressin to the bath, CCT Pd remained unchanged for 71 +/- l0s (n = 9, 37 degree C) and 170 +/- 45 s (n = 4, 23 degree C); this was followed by a slow increase in Pd(TI/2 = 91 +/- 17 s, 37 degree C; 119 +/- 31 s, 23 degree C) to the new steady-state value. These results provide a new approach for study of transepithelial water transport in kidney tubules. Compared with 3H20 methods, the fluorescence method is superior in technical simplicity, time resolution, and accuracy. The improved time resolution is important for examination of the pre-steady-state kinetics of vasopressin-induced signalling events resulting in the hydroosmotic response.

Involvement of singlet oxygen in the phototoxicity mechanism for a metabolite of piroxicam.

It has been previously shown that a metabolite of piroxicam but not piroxicam itself causes phototoxicity to cells in vitro after exposure to UVA (320–400 nm) radiation. The phototoxicity mechanism for this metabolite, 2‐methyl‐4‐oxo‐2H‐l,2‐benzothiazine‐l,l‐dioxide (Compound I), was investigated. In vitro phototoxicity to human mononuclear cells was assayed using 0.5 mM Compound I and UVA radiation. The UVA fluence required for phototoxicity of Compound I was lower by a factor of 2‐3 in D2O buffer compared to H2O buffer. Superoxide dismutase and mannitol, which remove O2‐ and OH”, respectively, do not decrease the phototoxicity. The photodecomposition of Compound I was inhibited by sodium azide, enhanced by human serum albumin and unaffected by mannitol. Stable photoproducts of Compound I were not toxic to the cells. The quantum yield of singlet oxygen based on its emission at 1270 nm was 0.19 and 0.35 for Compound I and s2 ± 10‐3 and 10‐2 for piroxicam in D2O and C6H6, respectively. While the extremely low quantum yield for singlet oxygen from piroxicam appears to account for its lack of phototoxicity, the phototoxicity mechanism for its metabolite, Compound I, most likely does involve singlet oxygen.

Photo-oxidation effects on beta-hydroxybutyrate dehydrogenase: studies of membrane fragments and intact mitochondria.

The influence of singlet oxygen (1O2), generated by red light irradiation of oxygenated suspensions containing aluminium phthalocyanine sulphonate, on the membrane bound enzyme β‐hydroxybutyrate dehydrogenase was investigated. The inactivation rates were measured using a spectrophotometry assay which involves disruption of the mitochondria. A novel NMR assay was also used to measure the activity of the enzyme in intact mitochondria. Relatively high inactivation rates of around 109M−1 s−1 were observed in H2O buffer, and rates in D2O were a factor of 1.7 faster. Significant differences in enzyme inactivation rates by 1O2 were observed, not only between disrupted and intact mitochondria but also between the NMR assay results and the spectrophotometric assay results. The results indicate the value of a specific assay which does not require the disruption of the biological system.

Kinetics of acid-catalyzed hydration in aqueous solution of 1-methoxy- and 1-methylthio-2-phenylethyne and some related acetylenes

The rates of conversion of C6H5C≡COCH3 to C6H5CH2CO2CH3 were measured in dilute HClO4/H2O, DCIO4/D2O, and H3PO4–H2PO2−/H2O buffer solutions, and the rates of conversion of C6H5C≡CSCH3 to C6H5CH2COSCH3, C6H5C≡CH to C6H5COCH3, 2,4,6-(CH3)3C6H2C≡CH to 2,4,6-(CH3)3C6H2COCH3, and p-CH3OC6H4C≡CCH3 to p-CH3OC6H4COCH2CH3 were measured in concentrated HClO4/H2O solutions, all at 25 °C. The reaction of C6H5C≡COCH3 showed general acid catalysis and gave the isotope effect [Formula: see text], which indicates that it proceeds through rate-determining proton transfer from catalyst to substrate. The hydronium ion catalytic coefficient for this reaction is [Formula: see text], and those for the other four, in the order given above, are [Formula: see text], and 8.5 × 10−6 M−1 s−1. Relative reactivities based on these rate constants are discussed.

Solution structure of a short DNA fragment studied by neutron scattering.

The solution structure of a DNA fragment of 130 base pairs and known sequence has been investigated by neutron small-angle scattering. In 0.1 M NaCl, the overall structure of the DNA fragment which contains the strong promoter A1 of the Escherichia coli phage T7 agrees with that expected for B-DNA. The neutron scattering curve is well fitted by that of a rigid rod with a length of 44 nm and a diameter of 2 nm. The results were confirmed by quasi-elastic light scattering and analytical centrifugation. The neutron measurements in H2O and D2O buffer reveal a cross-sectional inhomogeneity not detected by X-ray small-angle scattering. This inhomogeneity is caused by the hydration layer around the DNA core and not by the helical structure. The primary solvent shell has a density increased by at least 4-9% compared to bulk water.