pH-dependent Absorbance Research Articles

Colorimetric pH-sensing of artificial gastric fluid using naphthalimide-based CH acids

In this study, we introduce novel colorimetric pH-sensing probes based on naphthalimide malonate derivatives. These probes were synthesized by reacting 4-bromo-1,8-naphthalimide with various malonates, including malononitrile, ethyl cyanoacetate, and diethyl malonate. Each derivative exhibited distinct pH-sensing characteristics due to their differing CH acidities. The malononitrile-based probe, NPI-N2, demonstrated pronounced chromogenic pH-signaling behavior, transitioning from colorless to red-violet, accompanied by a decrease in fluorescence intensity. Notably, NPI-N2 retained its pH-sensing capability in the presence of common metal ions, anions, and pepsin, a key component of gastric fluid. The pKa of NPI-N2 was determined to be 3.08 through pH-dependent absorbance curve fitting. To modulate the pH-sensing range, ester-nitrile (NPI-EN) and diethyl ester (NPI-E2) subunits were incorporated into the naphthalimide framework, resulting in increased pKa values of 6.73 and 10.76, respectively. The pH-signaling mechanism of NPI-N2 was elucidated by 1H and 13C NMR spectroscopy, revealing deprotonation of the malononitrile moiety and subsequent resonance extension through the naphthalimide structure. To facilitate practical pH determination, NPI-N2 was integrated into a paper-based test strip, enabling convenient and reliable pH measurement of artificial gastric fluid.

Development of pH‐Sensitive Film for Detection of Implant Infection via Ultrasound Luminescent Chemical Imaging

AbstractA new hybrid ultrasound luminescent chemical imaging technique is described along with a pH sensor to image chemical concentrations at the surface of implanted medical devices. The purpose is to detect and study local biochemistry during infection. The sensor comprises a mechanoluminescent film (SrAl2O4:Eu, Dy microphosphors embedded in a biocompatible polymer film) and a pH indicator dye. A focused ultrasound beam generates green luminescence at the ultrasound focal point. By pulsing the ultrasound ON and OFF, the modulated luminescence can be distinguished from persistent luminescence, for high spatial resolution imaging. A red fluorescent dye and the pH indicator dye bromothymol blue are added to the coating to modulate the red‐light transmittance via pH dependent absorbance. Acidosis is observed as an increase in red luminescence intensity in spectroscopy and imaging. The films are sensitive to biologically relevant changes in pH (6.0–8.0) and can be imaged through optically scattering media to mimic tissue. The images have a knife edge spatial resolution of ≈3 mm through optically scattering phantoms, limited by the focused ultrasound spot size. This novel technique may permit the elucidation of implant infection at the implant surface and can be further developed for the measurement of other relevant chemical species in the future.

A novel flow-based procedure for automation of respirometric assays in soils

A flow-based strategy involving a gas-diffusion sampling probe was proposed for evaluating the respiration rate in soils. The amount of CO2 collected after a pre-defined time interval was proportional to the free CO2 released by the soil ecosystem. The 500-mL incubation flasks typically used for soil respirometric assays were adapted and a special cover was designed for connecting a tubular gas diffusion membrane, a fan, and a septum for adding the CO2(g) standards required for calibration. The method relied on the pH-dependent absorbance variations resulting from the CO2 collection. A 1.3mmolL−1 bromothymol blue solution (pH 7.0) acted as both acceptor and carrier streams. In order to widen the dynamical working range to 0.003–0.2mmol CO2, two analytical curves were obtained, each related to a different time interval for the CO2 collection. Kinetic curves related to CO2 release by the soil samples were straightforwardly attained. Repeatability and detection limit were estimated as 2.0% and 0.001mmol CO2 (n=10), and accuracy was assessed in relation to a recommended titrimetric procedure.

PH-dependent spectroscopy of tetracycline and its analogs.

Tetracyclines (TCs), broad spectrum antibiotics widely used in the prevention and treatment of infectious diseases, are amphoteric molecules containing several ionizable functional groups that exist predominantly as zwitterions at a given pH value. TCs are reported to undergo a wide variety of reactions at different pH values i.e. TCs form to anhydrotetracyclines at low pH, 4-epitetracyclines at pH 3-5 and isotetracyclines at high pH values. The pH-dependent absorbance and emission properties of tetracycline and its 10 analogs (4-epitetracycline, doxycyline, oxytetracycline, chlortetracycline, 4-epichlortetracycline, isochlortetracycline, methacycline, rolitetracycline, minocycline, and demeclocycline) were investigated and reported in this paper. The main focus of the study was on the pH dependent transformation of epichlortetracycline, chlortetracycline and isotetracycline at basic pH. Absorption, emission and time resolved spectroscopy were used to determine the behavior of the three TC derivatives at this condition. Increasing the buffer's ionic concentration leads to faster transformation to iCTC. A pH dependent transformation of CTC to iCTC was observed and the lifetimes of CTC and iCTC were determined to be 3.0 and 5.89 ns respectively. The distribution factor of CTC to iCTC at basic pH was also reported for the first time.

PH-dependent absorbance of a centrifuged rhodamine B-TiO2 suspension, and a practical filtration method with minimized dye loss in a dye decoloration test

Although the centrifugation of a rhodamine B (RhB)-TiO2 suspension cannot separate TiO2 particles completely, the UV/Vis absorbance of the residual TiO2 in the centrifuged suspension, under neutral and basic conditions, is not fully revealed under the presence of RhB molecules. Therefore, in a dye decoloration test, the centrifuged suspension can be used for estimating the concentration of RhB molecules based on the RhB absorbance peak in a Vis light regime. From acidic suspensions, however, the elimination of TiO2 is so difficult that the centrifuged suspension results in a significant shift of the baseline of the RhB absorbance peak. An additional use of a microfilter removes not only the residual TiO2, but also a part of the dye. A practical recipe for the successful microfiltration of TiO2 powder with a minimal loss of dye from the acidic suspension has been proposed and its feasibility has been validated.

Properties of the Thioredoxin Fold Superfamily Are Modulated by a Single Amino Acid Residue

The ubiquitous thioredoxin fold proteins catalyze oxidation, reduction, or disulfide exchange reactions depending on their redox properties. They also play vital roles in protein folding, redox control, and disease. Here, we have shown that a single residue strongly modifies both the redox properties of thioredoxin fold proteins and their ability to interact with substrates. This residue is adjacent in three-dimensional space to the characteristic CXXC active site motif of thioredoxin fold proteins but distant in sequence. This residue is just N-terminal to the conservative cis-proline. It is isoleucine 75 in the case of thioredoxin. Our findings support the conclusion that a very small percentage of the amino acid residues of thioredoxin-related proteins are capable of dictating the functions of these proteins.

Novel chemoselective linkage chemistry toward controlled loading of ligands to proteins through in situ real-time quantification of conjugate formation.

'Linkage chemistry', which encompasses the science of chemical attachment of a ligand molecule to a carrier moiety, plays a crucial role in a wide range of biochemical and biophysical disciplines. In particular, the production of synthetic vaccines, where quality assurance criteria are an essential part of the approvals procedure for development of medicines, is reliant upon reproducible linkage chemistries. Herein, we describe novel 2-hydroxybenzaldehyde-based quaternary amine containing chemoselective linkers that provide a simple and robust linkage process that overcomes the deficiencies present in state-of-the-art linkage chemistries. The 2-hydroxybenzaldehyde groups undergo a pH-dependent absorbance change that enabled its nondestructive quantification, even when covalently attached to a wide range of proteins. Additionally, formation of a hydrazone bond between the benzaldehyde group and a range of ligand hydrazides resulted in a second reversible absorbance change enabling the forward (ligand loading) and reverse (ligand release for analysis) reactions of ligand-loaded proteins to be monitored in situ and quantified in real time. Incorporation of the quaternary amine moiety into our improved linkage chemistries was found to increase the relative solubility of protein conjugates and enabled significantly higher loading of proteins with linker and subsequent ligands, while retaining aqueous solubility, when compared to standard methods.

Protonation equilibrium and lipophilicity of olamufloxacin (HSR-903), a newly synthesized fluoroquinolone antibacterial

This study was performed to characterize the protonation equilibrium at the molecular level and pH-dependent lipophilicity of olamufloxacin. The deprotonation fraction of the carboxyl group as a function of pH was specifically calculated at the critical wavelength 294 nm, where UV pH-dependent absorbance of olamufloxacin was independent of the ionized state of the aminopyrrolidinyl amino group but heavily depended on that of the carboxyl moiety. Accordingly, micro-protonation equilibrium could be described using a nonlinear least-squares regression program MULTI. In contrast, macro-protonation equilibrium was depicted at most wavelengths where olamufloxacin absorbance was influenced by ionized states of both proton-binding groups, results coinciding with the former. Furthermore, distribution features of four microspecies in aqueous phase were assessed. The apparent partition coefficient versus pH profile of olamufloxacin showed a parabolic curve in n-octanol/buffer system which reached peak near pH 8, agreeing with the above determined isoelectric point (p I). Ion-pair effect was observed for olamufloxacin under an acidic condition, eliciting experimental values higher than those theoretically calculated, which was similar to ciprofloxacin but not levofloxacin due to amino group type. Moreover, olamufloxacin was moderately lipophilic in comparison with other quinolones, with an apparent partition coefficient of 1.95 at pH 7.4.

Measurement of cytosolic, mitochondrial, and Golgi pH in single living cells with green fluorescent proteins.

Many cellular events depend on a tightly compartmentalized distribution of H+ ions across membrane-bound organelles. However, measurements of organelle pH in living cells have been scarce. Several mutants of the Aequorea victoria green fluorescent protein (GFP) displayed a pH-dependent absorbance and fluorescent emission, with apparent pKa values ranging from 6.15 (mutations F64L/S65T/H231L) and 6.4 (K26R/F64L/S65T/Y66W/N146I/M153T/ V163A/N164H/H231L) to a remarkable 7.1 (S65G/S72A/T203Y/H231L). We have targeted these GFPs to the cytosol plus nucleus, the medial/trans-Golgi by fusion with galactosyltransferase, and the mitochondrial matrix by using the targeting signal from subunit IV of cytochrome c oxidase. Cells in culture transfected with these cDNAs displayed the expected subcellular localization by light and electron microscopy and reported local pH that was calibrated in situ with ionophores. We monitored cytosolic and nuclear pH of HeLa cells, and mitochondrial matrix pH in HeLa cells and in rat neonatal cardiomyocytes. The pH of the medial/trans-Golgi was measured at steady-state (calibrated to be 6.58 in HeLa cells) and after various manipulations. These demonstrated that the Golgi membrane in intact cells is relatively permeable to H+, and that Cl- serves as a counter-ion for H+ transport and likely helps to maintain electroneutrality. The amenability to engineer GFPs to specific subcellular locations or tissue targets using gene fusion and transfer techniques should allow us to examine pH at sites previously inaccessible.

An absorbance-based fiber-optic sensor for CO2(aq) measurement in porewaters of sea floor sediments

Abstract We present results of the construction, testing and calibration of a sensor designed for in situ measurement of CO 2(aq) concentrations in porewaters of sea floor sediments. The sensor relies on pH-dependent absorbance changes of a dye-containing solution enclosed within a gas-permeable membrane. It is rugged enough to withstand insertion several centimeters in sediments without damage, and small enough to make measurements separated spatially by ≤ 2 mm. Response time at 20 °C is a few minutes. Calibration results imply that the sensor is capable of resolving differences in CO 2(aq) concentration of ± 8%, or about 2–3 × 10 −6 M, and is stable over time scales of several hours. Preliminary results from two sea floor locations in the equatorial Atlantic of depths 3300 and 4700 m are qualitatively consistent with simultaneous, adjacent pH electrode measurements. Sensor performance could be improved by adding a system for in situ reagent delivery, and by monitoring a non-absorbing wavelength in the dye absorbance spectrum to decrease dependence on the dye concentration of the internal solution.

Reactivity of Peroxynitric Acid (O2NOOH): A Pulse Radiolysis Study

Peroxynitrate (O2NOOH/O2NOO-) is formed within less than 2 ms after pulse irradiation of aerated solutions containing relatively low concentrations of formate and nitrate. The pKa for peroxynitric acid was determined to be 5.9 ± 0.1 both from the pH-dependent absorbance of the anion at 310 nm and from the dependence of the decay kinetics on pH. An absorption spectrum was measured for the anion giving emax(290) = 1500 ± 100 M-1 cm-1. This method of generation of peroxynitrate is very useful for studying the mechanism of the oxidation of various substrates by peroxynitrate. The oxidation by peroxynitrate can take place either directly or indirectly. In the direct oxidation pathway, the reaction is first order in peroxynitrate and first order in the substrate, whereas in the indirect oxidation pathway, the reaction is zero order in the substrate. In both cases, the observed rate constants are highly pH-dependent. The results show that the direct oxidation pathway takes place through O2NOOH. We suggest that t...

Local electrostatic potentials in pyridoxal phosphate labelled horse heart cytochrome c

The present work shows the application of an optical label pyridoxal phosphate (PLP) for the experimental determination of local electrostatic potentials in singly substituted cytochromes c modified by pyridoxal phosphate at Lys 79 (PLP-Lys-79-cyt.c) or at Lys 86 (PLP-Lys-86-cyt.c). PLP has also been used to calculate the p K a values of all ionizable groups and the electrostatic potentials in the modified proteins and to analyse their properties. The experimental p K a values for the pyridine nitrogen and phenolic hydroxyl of the bound label were obtained from pH-dependent absorbance and fluorescence measurements, as follows: in PLP-Lys-79-cyt.c for pyridine nitrogen 4.5 (absorbance) and 5.8 (fluorescence), for phenolic hydroxyl 8.3 (absorbance) and 8.5 (fluorescence). The differences between absorbance and fluorescence data are related to differences in the behaviour of the bound label in the ground and excited electronic states and to intermolecular charge-charge interactions. Molecular modelling was used to generate the atomic co-ordinates of the PLP-modified horse heart cytochrome c necessary for the theoretical calculations of the p K a values and electrostatic potentials.

Thermodynamic and kinetic studies of formation mechanisms of iron(III) complexes with N α-salicyl- l-alaninehydroxamic acid, l-alaninehydroxamic acid and N α-salicyl- l-alanine: role of hydroxamate group on enhancement of the stability

The formation kinetics and the thermodynamic stability of iron(III) complexes with a new tetradentate ligand, N α-salicyl- l-alaninehydroxamic acid ( 1, H 2slalh), have been investigated by the use of UV-vis and stopped-flow spectrophotometric methods. Two types of mono(ligand)iron(III) complex species of 1 are found to exist under both conditions of the metal ion concentration in large excess over the ligand and vice versa, by comparing the pH-dependent absorbance change behaviour of the iron(III) complex of 1 with that of the iron(III) complexes of l-alaninehydroxamic acid ( 2, Halh) and N α-salicyl- l-alanine ( 3, H 2slal), i.e. Fe(H 2slalh) 3+, which takes the coordination of the hydroxamate moiety below pH 1.8, and Fe(slalh) +, which takes the quadridentate coordination above pH 1.8. The complex formation reaction proceeds through one pathway of Fe 3+ + H 3slalh + ( k 33 I) for Fe(H 2slalh) 3+ and two pathways of Fe 3+ + H 3slalh + ( k 33 II) and FeOH 2+ + H 3slalh + ( k 23 II) for Fe(slalh) +. The mechanism of the complex formation was explained by the slow coordination of the carbonyl oxygen to iron(III) for Fe(H 2slalh) 3+ [and also Fe(Halh) 3+] and of the phenolato oxygen for Fe(slalh) + [and also Fe(slal) +], followed by the rapid donations of other coordinating atoms. The stability constants are calculated to be log β 11 = 10.29 ± 0.09 mol −1 dm 3 for Fe(H 2slalh) 3+ and 23.17 ± 0.18 mol −1 dm 3 for Fe(slalh) +, respectively. The introduction of the hydroxamate moiety to the carboxylate group of 3 increases the affinity of the ligand to iron(III), which indicates that the combination of hydroxamic acid is necessary to design a new type of siderophore based on the salicylamino acid ligand.

Ultraviolet-visible transient spectroscopy of bacteriorhodopsin mutants. Evidence for two forms of tyrosine-185—-phenylalanine.

The photocycle kinetics of the bacteriorhodopsin mutant Tyr-185----Phe has been investigated by UV-visible transient spectroscopy. Flash-induced spectral changes were measured from 100 ns to 500 ms using a gated optical multichannel analyzer on protein samples that were reconstituted in vesicles with Halobacterium halobium lipids. Tyr-185----Phe exhibits a pH-dependent absorbance spectrum reflecting contributions from two different species. At pH 6, the dominant photocycling species has a lambda max near 610 nm although the absorption maximum of light-adapted Tyr-185----Phe is at 581 nm. This red-shifted species does not form any M-like intermediate and undergoes a photocycle similar to that observed for deionized blue membrane. At pH 8, the dominant photoactive form exhibits a lambda max near 550 nm. This purple species, which is blue shifted 20 nm relative to wild-type bacteriorhodopsin, exhibits a photocycle similar to the wild type. However, M formation occurs in 8 microseconds, approximately three times faster than wild-type bacteriorhodopsin at pH 8. In addition, an unusually long lived intermediate absorbing at 610 nm is observed at high pH. In the UV region, a broad band near 300-310 nm is absent in the mutant relative to wild type, consistent with earlier measurements made at low temperature which suggest that Tyr-185 undergoes a change in protonation. Steady-state proton pumping action spectra indicate that the 550 nm species does transport protons but that the blue species is inactive. These results are discussed in terms of a model that hypothesizes that Tyr-185 is located close to the bacteriorhodopsin chromophore and stabilizes the interaction of helices F and G through formation of a polarizable bond with Asp-212.

Dissociation constants for dihydrofolic acid and dihydrobiopterin and implications for mechanistic models for dihydrofolate reductase

The dissociation constants (pKa) for the pteridine ring system of dihydrofolate (H2folate) have been redetermined, and those for dihydrobiopterin (H2biopterin) have been determined. Determination of the pKa for N5 of H2folate is complicated by the low solubility and instability of H2folate at pH 2-4, and other complicating factors. The initial rate of absorbance change due to degradation is a maximum at pH 2.5, and the products depend on the oxygen concentration: under aerobic conditions, (p-aminobenzoyl)glutamic acid and 7,8-dihydropterin-6-carboxaldehyde are major products. H2Biopterin is much more soluble and more stable at low pH. For protonation of N5, the pKa is 2.56 +/- 0.01 for H2biopterin and 2.59 +/- 0.03 for H2folic acid. Spectrophotometric determination of the pKa for the N3-O4 amide group of H2folate is subject to serious errors when a wavelength between 220 and 235 nm is used. These errors arise from the pH-dependent absorbance of mercaptoethanol often present in the preparation. The amide group has a pKa of 10.41 +/- 0.04 in H2biopterin and 10.85 +/- 0.04 in H2folate. The redetermined value for the pKa of N5 of H2folate has implications for mechanistic models for dihydrofolate reductase, and revised kinetic constants have been calculated for one model.

Regulation of cytoplasmic pH of cultured bovine corneal endothelial cells in the absence and presence of bicarbonate.

Intracellular pH (pHi) in confluent monolayers of cultured bovine corneal endothelial cells was determined using the pH-dependent absorbance of intracellularly trapped 5(and 6)carboxy-4',5'-dimethylfluorescein. Steady-state pH was 7.05 +/- 0.1 in the nominal absence of bicarbonate, and 7.15 +/- 0.1 in the presence of 28 mM HCO3-/5% CO2. Following an acid load imposed by a NH4Cl prepulse, pHi was regulated in the absence of HCO3- by a Na+-dependent process inhibitable to a large extent by 1 mM amiloride and 0.1 mM dimethylamiloride. In the presence of 28 mM HCO3-/5% CO2, this regulation was still dependent on Na+, but the inhibitory potency of amiloride was less. DIDS (1 mM) partially inhibited this regulation in the presence, but not in the absence of bicarbonate. With cells pretreated with DIDS, amiloride was as effective in inhibiting recovery from acid load as in the absence of HCO3-. The presence of intracellular Cl- did not appreciably affect this recovery, which was still sensitive to DIDS in the absence of Cl-. Removal of extracellular Na+ led to a fall of pHi, which was greatly attenuated in the absence of HCO3-. This acidification was largely reduced by 1 mM DIDS, but not by amiloride. Cl removal led to an intracellular alkalinization in the presence of HCO3-. The presence of a Cl-/HCO3- exchanger was supported by demonstrating DIDS-sensitive 36Cl- uptake into confluent cell monolayers. Thus, bovine corneal endothelial cells express three processes involved in intracellular pH regulation: an amiloride-sensitive Na+/H+ antiport, a Na+-HCO3- symport and a Cl-/HCO3- exchange, the latter two being DIDS sensitive.

THE EFFECT OF pH ON THE ABSORPTION AND FLUORESCENCE SPECTRA OF SANGUINARINE

Abstract— The influence of pH in the range 1.0 to 13.0 on the structure of sanguinarine has been investigated by spectrophotometric and spectrofluorimetric measurements. The data on absorption maxima, molar extinction coefficient, fluorescence emission maxima, relative fluorescence intensity and fluorescence quantum yield of the sanguinarine under various pH are presented. It is suggested that the pH dependent absorbance and fluorescence property of sanguinarine is due to the formation of the carbinolamine by hydroxylation at C‐6 at alkaline pH.

Effect of antibiotics on the dynamics of color change in Ureaplasma urealyticum cultures.

Color change of pH indicators in broth medium is commonly used to quantify growth of ureaplasmas. These organisms differ from other members of the Mollicutes by their ability to hydrolyze urea to CO2 and NH3. This study describes a method which continuously monitors color change in ureaplasmal broth cultures. Using this technique we found: (i) there was a pH-dependent absorbance at 554 nm in ureaplasmal broth medium containing phenol red, (ii) a sigmoidal-shaped color changing curve (absorbance at 554 nm versus time) was produced by metabolizing organisms whereas a linear curve was generated by antibiotic-inhibited ureaplasmas, and (iii) the minimum cell density which elicited a growth-inhibited color change was 1.25 x 10(4) colony-forming units per ml. Other have shown that apparently dead ureaplasmas can cause a color change in broth media. This color change is probably due to the presence of an active urease. This study graphically and quantitatively assesses growth-inhibited color change.