Analysis of dose-dependent antibody titration curves

Several types of dose-response titration curves were considered. It was demonstrated that the use of the so-called coordinates of dilution suggested earlier by us allows one to analyze the titration curves, obtained either by ELISA, or by agglutination. Theoretical curves, obtained by the developed theory are very similar to those obtained in experiments. It was shown, that the analysis of the titration curves could give important information concerning antibody-blocking factors in titration sera or other samples of studied antibodies.     

1H-NMR investigation of yeast cytochrome c. Interaction with the corresponding specific reductase (L-lactate cytochrome)

1H-NMR spectroscopy has been used to study the modifications of certain characteristic resonances of the Hansenula anomala yeast cytochrome c on binding to its specific reductase (flavocytochrome b2) or to the isolated cytochrome domain obtained from the entire molecule. Normal titration curves are observed for the resonances at 37.8 ppm assigned to heme c methyl 8 and at 19.4 ppm, line of cytochrome b2 spectrum. In contrast, the shifts near 3.2 and 3.4 ppm for trimethyl-lysine resonances of this cytochrome c present abnormal titration curves, saturation being apparently reached at low molar (cytochrome b2)/(cytochrome c) ratio. An interpretation is proposed in terms of shifts due to local conformational transitions induced by reductase binding but not rapidly reversible upon dissociation.     

Nuclear magnetic resonance titration curves of histidine ring protons. A direct assignment of the resonances of the active site histidine residues of ribonuclease.

One of the four titrating histidine ring C-2 proton resonances of bovine pancreatic ribonuclease has been assigned to histidine residue 12. This was accomplished by a direct comparison of the rate of tritium incorporation into position C-2 of histidine 12 of S-peptide (residues 1 to 20) derived from ribonuclease S, with the rates of deuterium exchange of the four histidine C-2 proton resonances of ribonuclease S under the same experimental conditions. The same assignment was obtained by a comparison of the NMR titration curves of ribonuclease S, the noncovalent complex of S-peptide and S-protein (residues 21 to 124) with the results for the recombined complex in which position C-2 of histidine 12 was fully deuterated. The second active site histidine resonance was assigned to histidine residue 119 by consideration of the NMR titration results fro carboxymethylated histidines and 1-carboxymethylhistidine 119 ribonuclease. This assignment is a reversal of that originally reported, and has important implications for the interpretation of NMR titration data of ribonuclease.     

Continuum electrostatic analysis of irregular ionization and proton allocation in proteins.

Irregular (nonsigmoidal) ionization behavior of titratable groups in proteins is analyzed theoretically, using a computational algorithm designed to count explicitly for tautomers of titratable groups and different locations of polar hydrogens. On the basis of calculations for different model systems (acid-acid, base-base, acid-base pairs, and cluster of three strongly interacting groups), it is demonstrated that the pK values, extracted from nonsigmoidal titration curves by fitting to a sum of Henderson-Hasselbalch equations, do not describe the ionization equilibrium correctly. The conditions for observation of irregular titration curves are derived analytically for the case of arbitrary couple of interacting ionizable groups. A possible relation between irregularly shaped titration curves and tautomerization is also illustrated. The protonation-deprotonation equilibrium of Asp76 in ribonuclease T1 is shown to be coupled to dipole reorientation of a water molecule bound at the protein-solvent interface. This finding provides a new interpretation of the experimentally observed chemical shift of this residue.     

Electrophoretic data in designing strategies for purification and identification of a highly polymorphic bacterial esterase

We have purified a bacterial enzyme, designated esterase M, by tailoring an efficient and rapid strategy with information derived from titration curves of proteins in crude extract. The pH-dependent stability of the enzyme activity observed by titration pattern allowed an acidic pH treatment of extract and a cationic exchange chromatography at pH 4.1. These two steps were followed by an anionic exchange chromatography and a preparative electrophoresis. Thus, the enzyme was purified about 2000-fold within two days with a recovery of 13.3%. The electrophoretic variants of esterase M were investigated for their molecular relationship through the specific effect of antibodies on esterase electrophoretic pattern (immunosubtractive electrophoresis) which is applicable to large series of samples. By this process, we have demonstrated the presence of common antigenic determinants among the electromorphs of esterase M produced by the three species of motile Aeromonas.     

On the Hill plot of NMR data for titration of protein residues

The Hill plots of NMR titration data for protein residues disclose more clearly than the usual titration curves the presence of multiple weak perturbations originating from other titratable groups, and should be used whenever the conventional curve fitting is poor. For a quantitative interpretation, we derive here expressions for the Hill equation and the Hill coefficient when the titration of the observed group is perturbed by more than one titratable group. When the generalized Hill equation is fitted to the data, values of the interaction parameters between the observed group and the others are extracted provided that there are no mutual interactions between the latter groups. The method is applied to the titration data of two histidyl residues of l-arginine phosphotransferase (E.C. 2.7.3.3.) in the transition state analogue complex (enzyme-Mg²⁺-ADP-NOsk3/–l-Arg). From the Hill plots, interactions with three titratable groups are disclosed for both residues, and the fitting with the Hill equation reveals that they experience perturbations from the same three groups. Microscopic pK values are obtained for all the involved groups, indicating large changes (up to 3 pH units) upon protonation of the interacting groups. As compared to the conventional fitting procedure, the use and fitting of Hill plots yields from NMR data more information on the neighbourhood of enzyme residues and on the changes intervening therein through the steps involved in the catalysis.     

THE VALENCE OF CORPUSCULAR PROTEINS

By the use of two extreme models: a hydrated sphere and an unhydrated rod the valence (net charge) of corpuscular proteins can be successfully calculated from electric mobility data by the Debye-Hückel theory (modified to include the effect of the ions in the ion atmosphere) in conjunction with the electrophoretic theory of Henry. As pointed out by Abramson, this permits a comparison with values for the valence from titration data. Electrometric titration measurements of serum albumin B (Kekwick) have been determined at several ionic strengths. These results, together with the available data in the literature for serum albumin B, egg albumin, and β-lactoglobulin have been used to compare values for the valence calculated from measurements of titration, electrophoresis, and membrane potentials. The results indicate that the usual interpretation of titration curves is open to serious question. By extrapolation of the titration data to zero ionic strength and protein concentration, there results an "intrinsic" net charge curve describing the binding of H⁺ (OH^-) ion alone. This curve agrees closely, in each case, with values of the valence calculated from mobility data (which in turn are in close accord with those estimated from membrane potential measurements). The experimental titration curves in the presence of appreciable quantities of ions and protein deviate widely from the ideal curve. It is suggested that, under these conditions, binding of undissociated acid (base) leads to erroneous values for the net charge. This binding would not affect the electrophoretic mobility. Values of the net charge obtained by the two extreme models from electrophoretic data are in agreement within 15 to 20 per cent. The agreement between the cylindrical model and the titration data is somewhat better in each case than with the sphere; i.e., this comparison enables a choice to be made between asymmetry and hydration in the interpretation of results from sedimentation and diffusion measurements on proteins. It is concluded that the proteins discussed here are somewhat asymmetric and also hydrated.     

Antigen-antibody binding in reverse micelles: interaction of monoclonal antibodies with a myelin basic protein peptide.

Reverse micelles can be used to mimic biological processes occurring at interfaces. To investigate antigen-antibody binding in a membrane-like environment, we first obtained Fab fragments from monoclonal antibodies against bovine myelin basic protein (MBP), an encephalitogenic protein. The binding of the fragments to a dansylated synthetic human MBP peptide gly(119)-gly(131), presenting sequence homologies with a viral protein, was measured in buffer and for the first time in reverse micelles of sodium bis(2-ethylhexyl) sulfosuccinate, in isooctane. Analysis of the fluorescence polarisation titration curves discloses that the Fab fragments in reverse micelles have retained the high affinity for the peptide found in buffer, and similar to that for intact MBP.     

A novel view of pH titration in biomolecules

When individual titratable sites in a molecule interact with each other, their pH titration can be considerably more complex than that of an independent site described by the classical Henderson-Hasselbalch equation. We propose a novel framework that decomposes any complex titration behavior into simple standard components. The approach maps the set of N interacting sites in the molecule onto a set of N independent, noninteracting quasi-sites, each characterized by a pK'(a) value. The titration curve of an individual site in the molecule is a weighted sum of Henderson-Hasselbalch curves corresponding to the quasi-sites. The total protonation curve is the unweighted sum of these Henderson-Hasselbalch curves. We show that pK'(a) values correspond to deprotonation constants available from methods that can be used to assess total proton uptake or release, and establish their connection to protonation curves of individual residues obtained by NMR or infrared spectroscopy. The new framework is tested on a small molecule diethylenetriaminepentaacetate (DTPA) exhibiting nonmonotonic titration curves, where it gives an excellent fit to experimental data. We demonstrate that the titration curve of a site in a group of interacting sites can be accurately reconstructed, if titration curves of the other sites are known. The application of the new framework to the protein rubredoxin demonstrates its usefulness in calculating and interpreting complicated titration curves.     

Reversible and irreversible changes in hydrogen ion titration curves of melanins.

Hydrogen ion titration curves obtained on melanins prepared and modified differently show recognizable differences. Melanin prepared with NaOH exhibits irreversible loss of functional groups in the base range once titrated acidic. Melanin prepared with NH4OH shows two main branches selected by incubation at an extreme pH for 24 h. Longer exposure reveals other curves but they disappear once a 24-h schedule of titration is resumed. The existence of two major pH branches appears consistent with the oxidation-reduction properties of melanins. These results demonstrate the ability to obtain a reproducible feature in the hydrogen ion titration curves. Thus it is finally possible to quantitate CO2 adsorbed to melanin in solution at basic pH.     

Proton titration curve of yeast iso-1-ferricytochrome c. Electrostatic and conformational effects of point mutations.

The proton titration curves of yeast iso-1-ferricytochrome c and selected point mutants of this protein have been determined between pH 3 and 11 at 10 and 25 degrees C with a computer-controlled titration system. Initial titration of the wild-type protein to acidic pH followed by subsequent titrations to alkaline and then acidic pH demonstrates hysteresis, with one more group (28.7) titrating between pH 11 and 3 than originally titrated (27.7) between pH 3 and 11. Initial titration to alkaline pH, however, resulted in observation of the same number of groups in both directions of titration (28.7 vs 28.6). At 10 degrees C, 7.5 fewer groups were found to titrate over the same range of pH. Titration curves obtained for six cytochrome c mutants modified at Arg-38, Phe-82, Tyr-48, and Tyr-67 were analyzed by subtraction of the corresponding titration curve for the wild-type protein to produce difference titration curves. In most cases, the effects of these mutations as revealed in the difference titration curves could be accounted for as either the result of introduction of an additional group titrating within this pH range, the result of a change in the pK of a titrating residue, and/or the result of a change in the pK for either the first acidic or the first alkaline protein conformational transition. In addition to demonstration of the electrostatic consequences of the mutations in cytochrome c studied here, this study establishes the general usefulness of precise proton titration curve analysis in the characterization of variant proteins produced through recombinant genetic techniques.     

Titration_DB: Storage and analysis of NMR‐monitored protein pH titration curves

NMR‐monitored pH titration experiments are routinely used to measure site‐specific protein pKa values. Accurate experimental pKa values are essential in dissecting enzyme catalysis, in studying the pH‐dependence of protein stability and ligand binding, in benchmarking pKa prediction algorithms, and ultimately in understanding electrostatic effects in proteins. However, due to the complex ways in which pH‐dependent electrostatic and structural changes manifest themselves in NMR spectra, reported apparent pKa values are often dependent on the way that NMR pH‐titration curves are analyzed. It is therefore important to retain the raw NMR spectroscopic data to allow for documentation and possible re‐interpretation. We have constructed a database of primary NMR pH‐titration data, which is accessible via a web interface. Here, we report statistics of the database contents and analyze the data with a global perspective to provide guidelines on best practice for fitting NMR titration curves. Titration_DB is available at http://enzyme.ucd.ie/Titration_DB . Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Stability of the folded-chain beta-structure of a homopolypeptide based on time-resolved potentiometric titrations

The standard free energy change of the unimolecular conversion between the folded-chain beta-structure and random coil of uncharged poly(S-carboxymethyl-L-cysteine) was evaluated from the potentiometric titration curves extrapolated to zero time to reduce the effect of aggregation which occurred at slower rates than chain folding for most degrees of ionization. To reduce the remaining contribution from the aggregation, the results obtained at finite concentrations were further extrapolated to zero polymer concentration. A value of -(400 +/- 100) cal/mol was obtained for a sample of chain length 630. From the titration curves at the aggregation equilibrium, the total free energy change of the whole solution associated with the same conversion was determined. The contribution made by aggregation to the stability was determined from the difference between these two quantities, which turned out to be comparable with that from the unimolecular conversion.     

The effect of discrete charges on the electrical properties of membranes. II

In this paper we calculate the hydrogen ion titration curves and associated surface potential for a lattice of discrete sites by numerically solving a titration equation constructed by expanding the partition function for the lattice in powers of the density of charged groups. The titration equations were calculated for several different lattice spacings and ionic strengths of the bathing solution. The results of applying this theory to several simple models of the membrane-solution interface is compared to the corresponding results obtained via the uniform-charge model.     

Antibodies against erythrocyte Ca2+-transport ATPase specifically inhibit the calmodulin-dependent fraction of the enzyme''s activity.

Antibodies against purified Ca2+-transport ATPase from human erythrocytes were raised in rabbits. Immunodiffusion experiments revealed that precipitating antibodies had been developed. The immunoglobulin fraction inhibited solely the calmodulin-dependent fraction of erythrocyte Ca2+-transport ATPase activity, whereas the basal (in the absence of added calmodulin) activity of the enzyme was not significantly affected by the antibodies. The antibodies produced similar doseresponse curves for the calmodulin- and the oleic acid-stimulated enzyme. However, the immunoglobulin fraction was considerably less effective in inhibiting Ca2+-transport ATPase activated by limited proteolysis. The results obtained with our antibodies are compatible with the interpretation that at least one subpopulation of the antibodies attacks the enzyme at or close to the calmodulin-binding site of the ATPase. The antibodies also inhibited the calmodulin-regulated Ca2+-transport ATPase from pig smooth-muscle plasma membrane, though with lower potency. However, the immunoglobulin fraction failed to suppress pig cardiac sarcoplasmicreticulum Ca2+-transport ATPase activity in the concentration range investigated. In addition, the activity of phosphodiesterase from rat brain, another enzyme modulated by calmodulin, was not at all affected by the immunoglobulin fraction.     

Temperature dependence of the hydrogen ion equilibria in poly(riboadenylic acid)

Hydrogen ion titration curves have been obtained for poly (riboadenylic acid) (poly A) at temperatures of 0–40°C, and ionic strengths of 0.001, 0.01, and 0.15. Where comparable, the data are in general agreement with those previously reported by other investigators. Correlations between the titration data and thermal denaturation curves have been obtained. Formation of a two-stranded helix is catalyzed by the uptake of protons by the adenine base. Partial protonation of the base is required for formation of the two-stranded helix, but under appropriate conditions it is stable at degrees of ionization less than 0.2. The degree of ionization required for formation of the two-stranded helix increases with temperature and decreases with ionic strength.     

A new ELISA for the three-spined stickleback (Gasterosteus aculeatus L.) spiggin, using antibodies against synthetic peptide

The aim of this study was to develop an enzyme-linked immunosorbent (ELISA) assay to quantify spiggin in the three-spined stickleback. Spiggin is a glue protein produced in the kidney of male three-spined stickleback under the control of androgens during the breeding period. Disturbances of spiggin production in male fish and abnormal induction of spiggin in female fish are considered as valuable biomarkers of exposure to (anti-)androgenic chemicals. Polyclonal antibodies against a peptide sequence of spiggin (HRD-16) were used and the specificity of the antibodies was verified by Western blotting and direct ELISA experiments. By using HRD-16 antibodies and spiggin standard preparation, a competitive ELISA was set-up and validated. This assay appears sensitive, with a detection limit of 0.5 U/mL, and specific, as shown by the competition curves, obtained by serial dilution of male and female kidney homogenates, that were parallel to the spiggin standard curves. The ability of the spiggin ELISA to quantify spiggin induction was achieved by exposing male and female three-spined sticklebacks to 0.1 and 1 microg/L of methyltestosterone. The results show a significant dose-dependent induction of spiggin in methyltestosterone-exposed female fish compared to controls.     

Protein interaction with macroporous silicas]

Potentiometric titration curves were obtained for macroporous silicas (MPS). The effect of the MPS structure on adsorption of various protein was studied. The maximal experimental and theoretical values of the adsorption capacity of a macroporous silica are presented.     

1H nuclear magnetic resonance titration curves and microenvironments of aromatic residues in bovine pancreatic ribonuclease A

The aromatic region of the NMR spectrum of bovine pancreatic ribonuclease A was analyzed in order to clarify the nature of the microenvironments surrounding the individual histidine, tyrosine, and phenylalanine residues and the interactions with inhibitors. The NMR titration curves of ring protons of six tyrosine and three phenylalanine residues as well as four histidine residues were determined at 37 degrees C between pH 1.5 and pH 11.5 under various conditions. The titration curves were analyzed on the basis of a scheme of a simple proton dissociation sequence and the most probable values were obtained for the macroscopic pK values and intrinsic chemical shifts. The microenvironments surrounding the residues and the effects of inhibitors are discussed on the basis of these results. Based on the titration curves of ring protons, the six tyrosine residues were classified into the following four groups: (1) titratable and different chemical shifts for C(delta) and C(epsilon) protons (two tyrosine residues), (2) titratable but similar chemical shifts for C(delta) and C(epsilon) protons (two tyrosine residues), (3) not titratable and different chemical shifts for C(delta) and C(epsilon) protons (one tyrosine residues), and (4) not titratable and similar chemical shifts for C(delta) and C(epsilon) protons (one tyrosine residue). The resonance signals of ring protons were tentatively assigned to tyrosine and phenylalanine residues. The NMR titration curves of His-48 ring protons were continuous in solution containing 0.2 M sodium acetate but were discontinuous in solution containing 0.3 M NaCl because the NMR signals disappeared at pH values between 5 and 6.5. The effects of addition of formate, acetate, propionate, and ethanol were investigated in order to elucidate the mechanism of the continuity of the titration curves of His-48 in the presence of acetate ion. The NMR signal of His-48 C(2) protons was observed at pH 6 in the presence of acetate and propionate ions but was not observed in the presence of formate ion or ethanol. This indicated that both the alkyl chain and the anionic carboxylate group are necessary for the continuity of the titration curves of His-48 ring protons. Based on the results, the mechanism of the effects of acetate ion is discussed.     

Potentiometric titration curves of oxidized and reduced horse heart cytochrome c

Potentiometric titration curves of oxidized and reduced horse heart cytochrome c in 0.15M KCl at 20°C have been obtained by timed titration (0.125–0.500 μmol/sec) from the isoionic points (pH 10.2–10.4) to pH 3 and back to the isoionic point. Computer-assisted (PROPHET) data acquisition and blank corrections give curves with good precision with a maximum standard deviation of 0.3 groups for an average error of 1%. The potentiometric titration curve of reduced cytochrome c is reversible within the precision of the method and for the pH range studied. The potentiometric curves for oxidized cytochrome c titrated upscale (pH 3–10) and downscale (pH 10–3) are not reversible. However, they show the same ionization behavior after the initial downscale titration. This is probably the result of a conformational change. Comparison of the data herein reported with the titration curves of oxidized cytochrome c already published by others indicates good agreement on the basis of a normalization of the concentration of protein or on the basis of 25 titrable groups between the acid end point and the isoionic pH. Titration of the 2 μmol imidazole in the upscale or downscale direction gives the correct analytical concentration and pK′ after correction for the solvent titration. Titration of reduced cytochrome c in the presence and absence of an additional equivalent of imidazole gave a difference titration curve, which indicates that a group on the protein shifts from pK′ 5.8 to pK′ 5.3 in the presence of imidazole. The pK′ of imidazole, in the presence of the protein, remains at a nearly normal value of 7.34.