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1.
Bobrovnik SA 《Ukrainski? biokhimicheski? zhurnal》2012,84(2):105-110
Different forms of titration curves obtained by ELISA for serum antibodies, monoclonal antibodies and polyreactive immunoglobulins were considered. A new interpretation of dose-dependent titration curves was suggested. It was shown that our interpretation of dose-dependent titration curves for antibodies which are presented in some biological liquids allow obtaining additional information about properties of the samples. This information was not obtained earlier because of the wrong understanding of the considered problem. 相似文献
2.
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. 相似文献
3.
Multilayer films of complexes of polycytidylic acid with dioctadecyldimetylammonium were obtained by the Langmuir-Blodgett method (LB films), and complexes of poly(C) with polycations (poly-L-lysisne, polyethyleneimine, polyallylamine) were obtained by the method of alternate adsorption (polyionic assembly) from solutions of oppositely charged polyelectrolytes on the solid carrier (SA films). It was shown that poly(C) exists in SA films in a single-stranded state irrespective of whether in the starting solution it occurred in the single-stranded nonprotonated or double-stranded protonated conformation. Conversely, in the LB film poly(C) preferred to be in a double protonated conformation. UV-spectra of water-insoluble LB and SA films at different pH values of surrounding water medium were investigated. Proton titration curves of poly(C) immobilized in LB films were obtained. The analysis of the shape of titration curves showed that the molecular-ionic transformation of poly(C) in LB films is accompanied by both the conformational transition of the polynucleotide and the molecular rearrangement in the whole film. Poly(C) was found to transform from the double- to single-stranded state and vice versa in the "deprotonation-protonation" cycle of LB film due to cooperative release/binding of hydrogen ions by cytosine bases. In contrast, poly(C) "protonation-deprotonation" in SA films occurred without conformational transitions of the polynucleotide. As opposed to poly(C) in solution a rather big hysteresis of forward and back titration curves was found for both types of multilayer films, indicating molecular rearrangements in films. The reason for the structural transformations of poly(C) upon fabrication of LB or SA films and the mechanism of molecular ionic transformations of poly(C) in films are discussed in terms of a simple model of ion exchange. An assumption about the nature of structural transformations of LB and SA films during their protonation-deprotonation is put forward. 相似文献
4.
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. 相似文献
5.
E V Aver'eva G Ia Kivman M N Markovich N F Shra?ber O T Pognoevski? 《Antibiotiki i khimioterapii͡a》1988,33(6):444-448
Simultaneous binding of two drugs to human serum albumin (HSA) was studied by flow microcalorimetry. The following drug pairs were used: sulfadimethoxine and cefazolin. Sulfadimethoxine and dicloxacillin, sulfadimethoxine and chlortetracycline. A procedure for estimating the calorimetric titration curves in competing binding of the drugs to the HSA homogeneous active site is described. Comparison of the theoretical and experimental titration curves enabled detection of the ligand competition for the biopolymer binding site. It was shown that sulfadimethoxine displaced cefazolin in the HSA active site, the nature of the HSA association with dicloxacillin and sulfadimethoxine was independent and binding of doxycycline or chlortetracycline to HSA had no influence on sulfadimethoxine interaction with protein. 相似文献
6.
Potentiometric titrations of poly(S-carboxymethyl-L -cysteine) and poly(S-carboxy-ethyl-L -cysteine) were carried out in aqueous sodium chloride solutions and in water. For samples of both polymers of high molecular weight, a new pattern was observed concerning the change of titration curve with time; the β-coil transition became sharper and the transition free energy increased by about 100 cal mole?1 as the equilibrium was approached. This suggests that equilibrium data were not obtained in most previous studies on the titration involving the β-coil transition. It also shows that the reversbility is not necessarily sufficient to confirm the equilibrium. Another pattern, which was previously observed, was also confirmed with a low molecular weight sample of poly(S-carboxymethytl-L -cysteine). The titration curves were shown to be insensitive to polymer concentration, even when aggregation or phase separation was present. The validity of the Gouy model to describe the titration curve of the β-structure was found to depend on molecular weight as well as on the nature of the side chain. 相似文献
7.
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. 相似文献
8.
Amperometric titration with iodine, a method devised by Kobayashi and Yoshida, was applied to various starch-type polysaccharides in order to elucidate the relation between the shape of titration curves and the structure of substances. Shapes of titration curves could be divided into 2 types, provisionally named the curves of amylose-type, and amylopectin-type, respectively.Amyloses and linear dextrins with the degree of polymerization (DP) above 28 gave curves of the former type, which exhibited segments with more or less low gradient in the former half of the titration.On the other hand, all of the branched polysaccharides, linear dextrins with DP below 25, Schardinger dextrins, and polyvinyl alcohol showed curves of the latter type, which rised linearly with added iodine.The mode of interaction with iodine was discussed for these polysaccharides together with some observations on the starch-iodine coloration. 相似文献
9.
Both rat liver and Escherichia coli rRNA in 0.1m-sodium chloride were titrated with acid or alkali over the range pH3-7 at approx. 0 degrees C. rRNA did not bind acid reversibly and hysteresis was observed, i.e. the plot of acid bound to rRNA against pH had the form of a loop showing that the amount of acid bound at a particular pH depended on the direction of the titration. Although the boundary curves were reproducibly followed on titration from pH7 to 3 and from pH3 to 7, points within the loop were ;scanned', e.g. by titration from pH7 to a point in the range pH3-4 followed by titration with alkali to pH7. It is inferred that the ;lag' in the release of certain bound protons is at least 1 pH unit, that at least about 9-15% of the titratable groups (adenine and cytosine residues) that are involved in this process and that the free energy dissipated in completing a cycle is approx. 4.2kJ/mol (1kcal/mol) of nucleotide involved in hysteresis. The interpretation of the ;scanning' curves was illustrated by means of a cycle of possible changes in the conformation of a hypothetical nucleotide sequence that allows formation of poly(A).poly(AalphaH(+))-like regions in acidic solutions. It is also inferred that the extent of ;hysteresis' might depend on the primary nucleotide sequence of rRNA as well as on secondary structure. 相似文献
10.
J E McGinness P R Crippa D S Kirkpatrick P H Proctor 《Physiological chemistry and physics》1979,11(3):217-223
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. 相似文献
11.
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. 相似文献
12.
Astrid R Klingen Elisa Bombarda G Matthias Ullmann 《Photochemical & photobiological sciences》2006,5(6):588-596
This paper presents a theoretical analysis of the titration behavior of strongly interacting titratable residues in proteins. Strongly interacting titratable residues exist in many proteins such as for instance bacteriorhodopsin, cytochrome c oxidase, cytochrome bc(1), or the photosynthetic reaction center. Strong interaction between titratable groups can lead to irregular titration behavior. We analyze under which circumstances titration curves can become irregular. We demonstrate that conformational flexibility alone can not lead to irregular titration behavior. Strong interaction between titratable groups is a necessary, but not sufficient condition for irregular titration curves. In addition, the two interacting groups also need to titrate in the same pH-range. These two conditions together lead to irregular titration curves. The mutation of a single residue within a cluster of interacting titratable residues can influence the titration behavior of the other titratable residues in the cluster. We demonstrate this effect on a cluster of four interacting residues. This example underlines that mutational studies directed at identifying the role of a certain titratable residue in a cluster of interacting residues should always be accompanied by an analysis of the effect of the mutation on the titration behavior of the other residues. 相似文献
13.
S Iida 《Journal of biochemistry》1979,86(3):733-743
Structural changes of troponin C on calcium binding were studied by hydrogen ion titration, circular dichroism, and fluorescence measurements. The potentiometric titration curves in the carboxyl region are shifted towards lower pH with calcium binding. The intrinsic pK of the carboxyl groups at the calcium binding sites decreases by 0.8 pK unit on calcium binding; on the other hand, magnesium ions have little effect on the intrinsic pK of the carboxyl groups. The intrinsic pK of the imidazole group is not affected by calcium binding. The value of w, an electrostatic interaction factor, is identical for calcium-free and calcium-bound troponin C and is about half of the value calculated assuming a compact sphere. The results of difference titration on the calcium binding indicate that the pH of troponin C solution increases on addition of CaCl2 up to 2 mol of Ca2+ per mol of troponin C and then decreases on further addition of CaCl2. The pH increase is depressed in the presence of MgCl2, in the low pH region, or at high ionic strength. The pH increase is also observed on addition of MgCl2. The ellipticity at 222 nm was measured under the same conditions as the difference titration measurements, and the relation between the pH change and the conformational change of troponin C on calcium binding is discussed based on the results obtained. The number of calcium binding sites and the binding constants estimated by analysis of these difference titration curves were in agreement with the results of Potter and Gergely (22). No magnesium binding site was observed. The tyrosine fluorescence measurements indicated that the binding site near tyrosine-109 is one of the high affinity sites. 相似文献
14.
The stability constants of copper(II) complexes with some alpha-amino acids in dioxan-water mixtures
In this study, the overall stability constants of copper(II) complexes with some alpha-amino acids (glycine, dl-alanine, dl-valine, l-leucine, l-asparagine, l-glutamine) were determined by potentiometric titration in water, 25% dioxan-75% water, 35% dioxan-65% water, 50% dioxan-50% water, and 60% dioxan-40% water. The titrations were performed at 25 degrees C, under nitrogen atmosphere, and the ionic strength of the medium was maintained at 0.10 M by using sodium perchlorate. The formation curves of their complexes (n-p[L]) were obtained by means of the titration data. Then the stability constants were determined in relation to these curves. The mol ratio of copper(II) to alpha-amino acid was also determined and it was found that the complexes were CuL(2) type. Another important result obtained was that the tendency of amino acids to form complexes with copper(II) was greater in dioxan-water mixtures compared to water. 相似文献
15.
Several methods were employed for the preparation of salt-free trypsin samples which were used to determine the electrometric titration curves of the enzyme. These curves point to a maximum acid-binding capacity below pH 2. Stoichiometric analysis indicates the presence of 6 carboxyl groups per 10,000 g. of proteins, 1 imidazole group, and 13 hydroxyl-binding groups. Calcium has a specific effect on the titration curves by increasing the acidity of the carboxyl groups in the pH range 3.5 – 5. This effect is not shown by potassium, sodium, or even the bivalent magnesium ion. It is attributed to the formation of a specific complex between the enzyme and the calcium ions, involving the carboxyl groups of the protein. The equally specific protective effect of calcium on the self-digestion of trypsin can therefore be explained by assuming the formation of a complex which stabilizes the enzyme. 相似文献
16.
The binding of hydrogen ions to an acidic glycoprotein from bovine cortical bone 总被引:2,自引:2,他引:0
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The H+ ion dissociation of bone sialoprotein in 0·2m-sodium chloride at 25° was studied. The total content of carboxyl groups available for titration was calculated by comparing the titration curve with the titration curves of three model systems and by the use of analytical data. This comparison showed that 7·0 carboxyl groups/mol. do not participate in the titration, and it is proposed that these are aspartic acid or glutamic acid carboxyl groups present as amides; this is also indicated by titration of the sialoprotein after acid hydrolysis. The titration of carboxyl groups was found to agree well with the Linderstrøm-Lang equation for spherical macroions. 相似文献
17.
The Interaction of the Polyene Antibiotic Lucensomycin with Cholesterol in Erythrocyte Membranes and in Model Systems: I. A Fluorometric and Spectrophotometric Study 总被引:1,自引:1,他引:0
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The increase of fluorescence emission of lucensomycin occurring upon interaction with cholesterol, either as colloidal suspension or included in phospholipid micelles or in erythrocyte membranes, was described. Colloidal cholesterol differed from that contained in membranes by the kinetic behavior of its interaction with lucensomycin, the reaction being very slow in the former case, very fast in the latter one. Variations of optical density also occurred, though neither the kinetics nor the titration curves were superimposable on those obtained fluorometrically. The stoichiometry was, however, the same. 相似文献
18.
1H nuclear magnetic resonance titration curves and microenvironments of aromatic residues in bovine pancreatic ribonuclease A 总被引:1,自引:0,他引:1
M Tanokura 《Journal of biochemistry》1983,94(1):51-62
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. 相似文献
19.
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. 相似文献
20.
M A Thomas M A Delsuc J C Beloeil J Y Lallemand 《Biochemical and biophysical research communications》1987,145(3):1098-1104
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. 相似文献