Acid Dissociation Constant Values Research Articles

ACID-BASE PROPERTIES OF ALKYLENE­DIAMINETETRA (METHYLENEPHOSPHONIC) ACIDS

The acid-base characteristics of ethylenediaminetetra(methylenephosphonic) (edtmp), tetramethylenediaminetetra(methylenephosphonic) (tetdtmp) and pentamethylenediaminetetra(methylenephosphonic) (pendtmp) acids were investigated by means of pH-potentiometry and 31P NMR spectroscopy. Calculated and estimated values of acid dissociation constants (pK) for edtmp (рК1<2, рК2<2, рК3=3.07, рК4=5.17, рК5=6.52, рК6=7.97, рК7=9.65, рК8=10.94), for tetdtmp (рК1<2, рК2=4.48, рК3=5.34, рК4=6.13, рК5=6.66, рК6=10.08, рК7~11.4, рК8~11.5) and for pendtmp (рК1<2, рК2=4.22, рК3=5.56, рК4=6.16, рК5=7.66, рК6=10.99, рК7~11.5, рК8~11.6) were obtained. The values of the acid dissociation constants indicate a decrease in the acidity of phosphonic groups and an increase in the basicity of amino groups with increasing hydrocarbon chain length between the two nitrogen atoms in the edtmp, tetdtmp, and pendtmpseries. Based on the values of the calculated constants for the formation of anions of tetra­phosphonic acids, diagrams of the distribution of their equilibrium concentrations depending on pH were constructed, which indicate the state of acid-base equilibrium, the charge and the ratio of concentrations between anions with different degrees of protonation at certain pH values.
 According to the data of 31P NMR spectro­scopy, the formation of intramolecular hydrogen bonds between the deprotonated oxygen atoms of one amino(methylphosphonic) group with protonated nitrogen atom of another amino(methylphosphonic) group was established in the case of edtmp. In the case of pendtmp and tetdtmp, the formation of intermolecular hydrogen bonds between distant amino(me­thyl­phosphonic) groups is impossible due to the presence of steric hindrances.

Физико-химические свойства этилового эфира N'-тозилгидразида щавелевой кислоты

The results of a study of the physicochemical properties of N'-tosylhydrazide oxalic acid (ETH) ethyl ester are presented. The solubility of the reagent in ethanol, 0.1 mol/l KOH solution, toluene, chloroform, hexane, and water was studied by spectrophotometry, refractometry, and gravimetry. Based on the results obtained, it was shown that ETG can be used in flotation and extraction processes. Acid-base equilibria in reagent solutions were studied by spectrophotometric method. The obtained values of acid dissociation constants (pKa1=7.56 ± 0.14; pKa2=13.48 ± 0.22) prove that ETG is a weak dibasic acid. The hydrolytic stability of the reagent was studied by the spectrophotometric method. The results of the study showed that ETG solutions are quite stable in time in alkaline media: the degree of hydrolysis for two hours is 4.77%. The surface activity of the reagent was determined by the stalagmometric method. ETG has been found to be a surfactant.

Spectrophotometric determination of acid dissociation constants of some arylpropionic acids and arylacetic acids in acetonitrile-water binary mixtures at 25ºC

The acid dissociation constant of drug active compounds (arylpropionic and aryl acetic acids) were determined in acetonitrile and water binary mixtures (corresponding volume fractions of 0.40, 0.45, 0.50, and 0.55) by using a multi-wavelength spectrophotometric method. Drug active compounds, which were slightly soluble in water, were studied in these binary mixtures. The dissociation constants of drug active compounds are important in drug design studies and in any research of the biopharmaceutical and physicochemical properties of drugs. The STAR program was used for the determination of dissociation constants. The acidity constants of arylpropionic and aryl acetic acids were correlated with the Kamlet and Taft solvaatochromic parameters. Aqueous pKa values of these arylpropionic and aryl acetic acids were determined from pKa values obtained from acetonitrile and water binary mixtures with varying volume fractions. The studied drugs had a pKa value corresponding to base functional group. Results showed that the acid dissociation constant values of the drug active compounds increased with an increase in acetonitrile content in the medium.

Bacteria-Catalyzed Arginine Glycosylation in Pathogens and Host

In recent years, protein glycosylation in pathogenic bacteria has attracted more and more attention, and accumulating evidence indicated that this type of posttranslational modification is involved in many physiological processes. The NleB from several enteropathogenic bacteria species as well as SseK from Salmonella enterica are type III secretion system effectors, which have an atypical N-acetylglucosamine (N-GlcNAc) transferase activity that specifically modified a conserved arginine in TRADD, FADD, and RIPK1. NleB/SseKs GlcNAcylation of death domain proteins abrogates homotypic and heterotypic death receptors/adaptors interactions, thereby blocking an important antimicrobial host response. Interestingly, NleB/SseKs could also GlcNAcylate themselves, and self-GlcNAcylation of NleB, SseK1, and SseK3 are crucial for their biological activity during infection. In addition, EarP (EF-P specific arginine rhamnosyl transferase for Posttranslational activation) catalyzes arginine rhamnosylation of translation elongation factor P (EF-P). Importantly, this kind of N-linked protein glycosylation is not only important for EF-P dependent rescue of polyproline stalled ribosomes but also for pathogenicity in Pseudomonas aeruginosa and other clinically relevant bacteria. Glycosylation of arginine is unique because the guanidine group of arginine has a high acid dissociation constant value and representing an extremely poor nucleophile. Recently, the crystal structures of NleB, SseKs, EarP, arginine GlcNAcylated death domain-containing proteins, NleB/FADD-DD, and EarP/EF-P/dTDP-β-L-rhamnose were solved by our group and other groups, revealing the unique catalytic mechanisms. In this review, we provide detailed information about the currently known arginine glycosyltransferases and their potential catalytic mechanisms.

EXPLORING OF ACID-BASIC PROPERTIES, DEVELOPMENT AND VALIDATION OF THE METHOD OF QUANTITATIVE DETERMINATION OF ORIGINAL PHARMACEUTICAL SUBSTANCE

Introduction. The object of analysis was a newly synthesized organic substance, a derivative of 4-aminobenzoic acid and malonic ester – 4,4’-(propandiamido) sodium dibenzoate, which has an antisteatotic effect. For the quantitative determination of pharmaceutical substances, it is preferable to use titrimetric methods of analysis, since they are absolute and allow us to estimate the content of the active compound in the substance without using standard samples. Aim. The objective of this work was to develop and validate a quantitative determination method for 4.4’-(propandiamido) sodium dibenzoate. Materials and methods. The analyzed compound is a salt of a weak dibasic acid, which exhibits basic properties in an aqueous solution; therefore, for its quantitative determination, a direct acid-base titration method (acidimetry) was proposed. The method of quantitative determination of the active compound in the substance 4,4’-(propandiamido) sodium dibenzoate will be used in future studies for routine analysis, therefore a visual (indicator) method for determining the end point of the titration has been chosen. To prove the suitability of the developed method and the possibility of obtaining reliable results of the quantitative content of the active compound in the newly synthesized pharmaceutical substance the used analytical method was validated. Results and discussion. The study of the acid-base properties of the newly synthesized substance was carried out using the method of potentiometric titration, on the basis of which the values of the dissociation constants were calculated. Using spreadsheets, the pKa1 and pKa2 values were calculated using a titration model of a multi-basic acid, the pKa values of which are 7.2 and 5.0. The titration curve that was constructed from the experimental data and the curve constructed from the calculated values of the dissociation constants almost completely overlapped with each other. Based on the data obtained, a 0.1% water-alcohol solution of bromocresol green (blue) with pT=4.6 was selected for titration, the color transition interval of which falls into the titration jump of the analyte. The developed methodology was validated according to the requirements of the XIV edition State Pharmacopoeia of the Russian Federation by the following indicators: specificity, linearity, accuracy, precision (convergence and internal reproducibility), robustness, analytical domain. Conclusion. As a result of the performed studies, the values of acid dissociation constants of the newly synthesized pharmaceutical substance 4,4’-(propandiamido) sodium dibenzoate were determined, and a method for quantitative determination was developed and validated using acidbase titration.

Net Acid Excretion and Urinary Organic Anions in Idiopathic Uric Acid Nephrolithiasis.

Idiopathic uric acid nephrolithiasis, which is closely associated with obesity and the metabolic syndrome, is increasing in prevalence. Unduly acidic urine pH, the quintessential pathophysiologic feature of this disease, is in part explained by inadequate excretion of the principal urinary buffer ammonium. The role of net acid excretion in the pathogenesis of uric acid nephrolithiasis is incompletely understood. We compared acid-base parameters of patients with idiopathic uric acid nephrolithiasis with matched control subjects under controlled diets in an inpatient metabolic unit. Measurements included fasting blood and 24-hour urine chemistries and 24-hour urine metabolomic analysis. Comparisons between groups included analysis of covariance models controlling for urine pH or body mass index. Subjects with idiopathic uric acid nephrolithiasis had lower urine pH (5.5 versus 5.9; P<0.001) and higher net acid excretion (60 versus 43 mEq/24 h; P<0.001), with the excess H+ carried by nonammonium buffers. In all subjects, there was a positive relationship of net acid excretion with higher body mass index in spite of strictly controlled equivalent dietary acid intake. This relationship was most evident among control subjects (r=0.36; P=0.03). It was attenuated in patients with idiopathic uric acid nephrolithiasis whose net acid excretion remained fixedly high and ammonium excretion remained low relative to net acid excretion, resulting in low urine pH over a wide body mass index range. Urinary metabolomics was performed to attempt to identify excess organic acids presented to the kidney in idiopathic uric acid nephrolithiasis. Among the tricarboxylic acid cycle intermediates and amino acid and lipid metabolites analyzed, 26 organic anions with acid dissociation constants values in the range of urine pH showed greater protonation. However, protons carried by the identified organic acids did not entirely account for the higher titratable acidity seen in idiopathic uric acid nephrolithiasis. Higher acid load to the kidney, resulting in higher urinary net acid excretion, is an important factor in the pathogenesis of idiopathic uric acid nephrolithiasis.

A Benzooxazole‐Based Probe for the Sensitive Detection of Hydrogen Sulfide: Kinetic and Transition‐State Studies and In Vitro Application in HepG2 Cells

AbstractA benzooxazole based probe, 2‐[4‐(2, 4‐dinitrophenoxy)‐phenyl]‐benzooxazole has been synthesized and thoroughly characterized by mass, 1H‐NMR and FT‐IR spectroscopy. The probe exhibits sensitive and selective response towards H2S in DMSO:PBS buffer (6:4, v/v, pH 7.4) over the other reactive sulfur species especially the biological thiols like cysteine, homocysteine and glutathione. Due to thiolysis of dinitrophenyl ether, this probe displays turn‐on fluorescence response where the sensing mechanism follows inhibited photoinduced electron transfer (d‐PET) process. This thiolysis reaction proceeds through aromatic nucleophilic substitution (SNAr) type mechanism. Moreover, the transition state study shows that this process is exoergic with favorable energy barrier of 5.0 kcal/mol. The kinetic studies of the probe with HS– at various pH assist to evaluate the acid dissociation constant value (Ka1 =1.09 × 10−8 M−1) of H2S that illustrate a reasonable agreement with the literature value, Ka (9.5 x 10−8 M−1). Finally, this probe is cell permeable, non‐cytotoxic and suitable for intracellular cytoplasmic HS– sensing in live cells with a very low detection limit (51.8 nM).

Evaluation of Acid Dissociation Constants in DMSO and DMF by Quantum-Chemical Methods

We have calculated total electronic energies (E) and Gibbs energies (G) of a large number of acids and their anions in water, dimethylsulfoxide, and dimethylformamide using the hybrid B3LYP functional DFT method in the 6-31++G(d,p) basis set, taking into account the solvent effect by the conductor-like polarizable continuum model method. A linear correlation has been found between the experimental values of acid dissociation constants (pKa) of different nature and the difference between anion and acid E values, and between pKa and the difference between anion and acid G values. The obtained correlations allowed us to evaluate the pKa values of both inorganic and organic acids. Such an evaluation is of special importance for nonaqueous solvents as it is quite problematic to determine these dissociation constants.

Measurements of Thermodynamic Acid Dissociation Constants for Three HMG-CoA Reductase Inhibitors by using RPLC and Study of Validation in Pharmaceutical Tablets

Background: Present work was designed about the assignation of thermodynamic acid dissociation constants (pKa) of pravastatin, atorvastatin and rosuvastatin by using RPLC method. In the experiment, methanol-water binary mixture was chosen as hydroorganic solvent mixture. Method: Chromatographic study was performed with Nucleosil 300-5C4 column (5µm, 250x4.6 mm, I.D.) and the flow rate of the experiment was adjusted at 1 mL/min. The thermodynamic acid dissociation constant values and the intrinsic retention factors were estimated by use of a non-linear least squares fit of the data. The realibility of the RPLC methodology was investigated statistically using the difference between the experimental and theoretical results. Aqueous pKa values were also estimated with extrapolation by means of the Yasuda-Shedlovsky and mole fraction equations. Conclusion: Availability of present procedure in order to quantitatively determine the studied compounds was verified by the validation in suitability with the requirements laid down via ICH (International Conference on Harmonization) guidelines. Results of the analysis show that the method can be used for the estimation of studied drugs in pharmaceutical formulations, with great recovery, well precision and accuracy. Keywords: Statins, chromatographic retention, polarity parameter, thermodynamic dissociation constant, RPLC, method validation.

A computational analysis of S-(2-succino)cysteine sites in proteins

The adduction of fumaric acid to the sulfhydryl group of certain cysteine (Cys) residues in proteins via a Michael-like reaction leads to the formation of S-(2-succino)cysteine (2SC) sites. Although its role remains to be fully understood, this post-translational Cys modification (protein succination) has been implicated in the pathogenesis of diabetes/obesity and fumarate hydratase-related diseases. In this study, theoretical approaches to address sequence- and 3D-structure-based features possibly underlying the specificity of protein succination have been applied to perform the first analysis of the available data on the succinate proteome. A total of 182 succinated proteins, 205 modifiable, and 1750 non-modifiable sites have been examined. The rate of 2SC sites per protein ranged from 1 to 3, and the overall relative abundance of modifiable sites was 10.8%. Modifiable and non-modifiable sites were not distinguishable when the hydrophobicity of the Cys-flaking peptides, the acid dissociation constant value of the sulfhydryl groups, and the secondary structure of the Cys-containing segments were compared. By contrast, significant differences were determined when the accessibility of the sulphur atoms and the amino acid composition of the Cys-flaking peptides were analysed. Based on these findings, a sequence-based score function has been evaluated as a descriptor for Cys residues. In conclusion, our results indicate that modifiable and non-modifiable sites form heterogeneous subsets when features often discussed to describe Cys reactivity are examined. However, they also suggest that some differences exist, which may constitute the baseline for further investigations aimed at the development of predictive methods for 2SC sites in proteins.

Determination of Acid Dissociation Constant of Pravastatin under Degraded Conditions by Capillary Zone Electrophoresis.

The acid dissociation constant of pravastatin was determined under degraded conditions. Pravastatin was degraded in an acidic solution (pH = 2.0) for 5 h, and the degradation solution was subjected to the measurement of the effective electrophoretic mobility by capillary zone electrophoresis. Although the amount of pravastatin decreased by the acid degradation, its acid dissociation constant was successfully determined with the residual pravastatin through its effective electrophoretic mobility. The determined acid dissociation constant value agreed well with the one obtained with freshly prepared solution and with some reported values.

Extraction of mineral acids and lanthanum salts with binary extractants

Extraction of mineral acids with methyltrioctylammonium dialkylphosphinate was studied, and the extraction ability of acids was shown to decrease in the series HNO3 ≫ HBr > HCl, which corresponds to anion-exchange series for the initial salts of quaternary ammonium bases (QAB). Extraction of lanthanum bromide with methyltrioctylammonium dialkylphosphinate was studied, the composition of extracted compounds was established, and extraction constants were calculated. The extraction ability of dinonylnaphthalenesulfonate was found to be considerably lower than that of QAB dialkylphosphinate because of a larger value of acid dissociation constant for dinonylnaphthalenesulfonic acid. It was shown that lanthanum in systems with binary extractants can be back extracted from organic phase with dilute solutions of mineral acids.

RPLC determination of acid dissociation constants and quantitative estimation for sulfasalazine

Acid dissociation constant value (pK a) is key parameter for predicting the extent of the ionization of a drug molecule at different pH. The effect of the mobile phase composition on the acid dissociation constant values was studied by measuring the pK a values at different acetonitrile concentrations, ranging from 25 to 35 % (v/v). pK a values and limiting retention factors of SSZ were calculated chromatographically using equations derived. From calculated pK a values, the aqueous pK a values of SSZ were calculated by various approaches. Moreover, the correlation established between retention factors and the pH of the water-acetonitrile mobile phase was used to determine the optimum separation condition. Optimized condition was also validated with respect to system suitability, linearity, precision, accuracy, limit of detection and limit of quantitation, respectively. A Gemini NX C18 column (250 × 4.6 mm I.D., 5 µm particles) was used for all the determinations.

Open-cell Titration of Seawater for Alkalinity Measurements by Colorimetry Using Bromophenol Blue Combined with a Non-linear Least-squares Method

The open-cell titration of seawater was studied for alkalinity measurements by colorimetry. 1) The colorimetric pH of free hydrogen ion concentration, pH(F(ind)), was calculated from the ratio of the absorbances at 436 and 590 nm (R = (590nm)A/(436nm)A), along with the molar absorption coefficient ratios (e(1), e(2) and e(3)/e(2)) and a tentative acid dissociation constant value (pK(a(2))). 2) The perturbation of hydrogen ion was evaluated from the change in titration mass (Deltam). The total hydrogen ion concentration at m + Deltam, pH(T(at m+Deltam)), was calculated using pH(F(ind)) for a mass m and constants for sulfate (S(T)) and fluoride (F(T)). 3) The alkalinity (A(T)) was computed from the titrant mass (m + Deltam) and the corresponding pH(T(at m+Deltam)) through a non-linear least-squares approach using the pK(a(2)) value as a variable parameter. Seawater sample at 2000 m depth from the West Pacific was analyzed. The resulting A(T) (2420.92 +/- 3.35 micromol kg(-1)) was in good agreement with the A(T) measured by potentiometric electric force (2420.46 +/- 1.54 micromol kg(-1)). The resulting pK(a(2)) was 3.7037, in close proximity to that reported by King et al. (pK(a(2)) = 3.695).

Ion and pH Sensing with Colloidal Nanoparticles: Influence of Surface Charge on Sensing and Colloidal Properties

Ion sensors based on colloidal nanoparticles (NPs), either as actively ion-sensing NPs or as nanoscale carrier systems for organic ion-sensing fluorescent chelators typically require a charged surface in order to be colloidally stable. We demonstrate that this surface charge significantly impacts the ion binding and affects the read-out. Sensor read-out should be thus not determined by the bulk ion concentration, but by the local ion concentration in the nano-environment of the NP surface. We present a conclusive model corroborated by experimental data that reproduces the strong distance-dependence of the effect. The experimental data are based on the capability of tuning the distance of a pH-sensitive fluorophore to the surface of NPs in the nanometer (nm) range. This in turn allows for modification of the effective acid dissociation constant value (its logarithmic form, pK(a)) of analyte-sensitive fluorophores by tuning their distance to the underlying colloidal NPs.

Accuracy of the microsolvation–continuum approach in computing the pKa and the free energies of formation of phosphate species in aqueous solution

First principles density functional theory (Perdew-Burke-Ernzerhof) calculations have been used to compute the hydration properties, aqueous-phase acid dissociation constants (pK(a)) and Gibbs free energies of formation of small polyphosphates in aqueous solution. The effect of the hydrated environment has been simulated through a hybrid microsolvation-continuum approach, where the phosphate species are simulated as microsolvated solutes, while the remainder of the bulk solvent is treated as a dielectric continuum using the COSMO solvation model. The solvation free energies of orthophosphates and pyrophosphates have been computed applying monomer and cluster thermodynamic cycles, and using the geometries optimised in the gas-phase as well as in the COSMO environment. The results indicate that the simple polarisable continuum or microsolvation-continuum models are unable to compute accurate free energies of solvation for charged species like phosphates. The calculation of the pK(a) shows that the computed values of acid dissociation constants are critically dependent on the number of water molecules n(H(2)O) included in the hydrated phosphate clusters. The optimal number n(H(2)O) is determined from the minimum value of the "incremental" water binding free energy associated with the process of adding a water molecule to a micro-solvated phosphate species. Analysis of the effect of n(H(2)O) on the free energies of orthophosphate condensation reactions shows that can vary by tenths of kcal mol(-1), depending on the particular choice of n(H(2)O) for the monomeric and dimeric species. We discuss a methodology for the determination of n(H(2)O); for the orthophosphates the "incremental" binding energy approach is used to determine n(H(2)O), whereas for the polyphosphates the number of explicit water molecules is simply equal to the effective charge of these anions. The application of this method to compute the free energy of formation of pyro- and tri-phosphates gives generally good agreement with the available experimental data.

Characterisation of novel modified active carbons and marine algal biomass for the selective adsorption of lead

This paper discusses the sorption performance of novel materials for the removal of lead(II) and copper(II) from near-neutral aqueous solutions. Active carbons with surface heteroatoms of oxygen and phosphorus have been prepared. The surface functional groups display weakly acidic ion exchange characteristics. The optimum solution pH for maximum metal sorption is related to the pK values of the surface functional groups. In oxygenated active carbons, pK values are not distinct but can be obtained by describing proton binding to the heterogeneous adsorbent surface as a continuous proton affinity distribution. Information derived from zeta-potential measurements combined with knowledge of the pK distribution function and concentration of surface functional groups has been used to explain the selectivity of oxidised active carbons towards lead(II) in the presence of copper(II) from multi-metal bearing solutions. Marine algal-based biosorbents have been challenged with lead(II) and copper(II)-bearing wastewater. The weakly acidic carboxyl groups of structural polysaccharides present within the algal matrix display high sorption capacity for both metals. The negative surface charge of algal particles results in electrostatic interactions as well as coordination between metal species and the adsorbent surface. Proton affinity for the algal surface lowers the negative surface potential at pH values around 2. The surface functional groups in algae unlike those in oxidised active carbons may be represented by discrete acid-dissociation constant values. The influence of conformational differences in uronic-acid segments upon metal ion selectivity is discussed.

Application of Protonic Acid-Doped Silica Gels to Electric Double-Layer Capacitors

Silica gels doped with several protonic acids such as HClO4, H2SO4 and H3PO4 have been prepared by the sol-gel method and totally solid electric double-layer capacitors have been successfully fabricated using the highly proton-conductive silica gels as an electrolyte and activated carbon powder (ACP) hybridized with the silica gels as a polarizable electrode. It was found that the addition of HClO4, which had the highest value of acid dissociation constant among these three acids, most effectively increased the proton conductivity of the resultant acid-doped silica gels. Tablets of the HClO4-doped silica gels exhibited conductivities as high as 10−5–10−2 S cm−1 at room temperature in dry N2 atmosphere. One of the capacitors fabricated using the protonic acid-doped silica gels had a capacitance of 44 F/(gram of total ACP in the capacitor), which was comparable to those of conventional capacitors using liquid electrolytes.

Acid-base, hydration-dehydration and keto-enol equilibria in aqueous solutions of α-ketoacids: Study by spectroscopy, polarography and linear sweep voltammetry

In aqueous buffered solutions of oxalacetic and α-ketoglutaric acids and their esters, the most reliable values of acid dissociation constants K i were found using spectrophotometry, potentiometry, changes in wave-heights and shifts in polarographic half-wave potentials. Keto-enol equilibrium constants K E were obtained by spectrophotometry and polarography, and dehydration constants K d by linear sweep voltammetry. The combination of constants K i, K E, and K d in some cases enabled separation of specific dissociation constants of the enol, keto, and hydrated forms. These constants made possible calculation of the distribution of individual ionic forms at various pH values in dilute (10 −4 M) solutions, under conditions closer to those existing in biological systems and used in kinetic studies than information obtained by nuclear magnetic resonance in concentrated (greater than 1 M) solutions. In their acid-base and hydration-dehydration equilibria, structural effects operating in oxalacetic acid and its esters resemble those for α-ketoglutaric acid, the differences in their behavior in physical methods being predominantly due to the stability of enol forms of oxalacetic acid resulting from their β-ketoacid character. Whereas the monoanion −OOCCOCH 2COOH predominates between pH 5.1 and 12.3 in aqueous solutions of oxalacetic acid, the anion HOOCCOCH 2COO − is the reactive species in decarboxylation. A neglect of decarboxylation may account for some dubious results between pH 2 and 7 reported in the literature.

Direct measurement of the equilibrium between glutathione and dithiothreitol by high performance liquid chromatography

The equilibrium constant between reduced glutathione (GSH), oxidized glutathione (GSSG), reduced dithiothreitol (DTT SH SH), and oxidized dithiothreitol ▪ has been directly measured by high performance liquid chromatography analysis of equilibrium mixtures. The equilibrium constant at 25°C for the reaction GSSG + DTT SH SH ⇌ 2GSH + ▪ varies from approximately 200 M, below pH 8, to approximately 2800 M, above pH 11. The observed pH dependence is generally consistent with published values of acid dissociation constants of these thiols.