Titration Of Protein Research Articles

On the isoelectric point of human hair

The isoelectric point of human hair was found to fall in the 2.45–3.17 range. The acidic character of the hair surface appears to be due to ionic groups of oxyacids of sulfur. The Gilbert-Rideal theory, developed for the titration of fibrous proteins, was shown to hold well for human hair.

Spectrophotometric titration of muscle M-line proteins: Creatine kinase and the 165,000 dalton protein component

1. 1. The ionization of the tyrosine residues of rabbit skeletal muscle M-line creatine kinase and the 165,000 dalton protein component, as well as beef cardiac muscle creatine kinase, was measured spectrophotometrically as a function of pH. 2. 2. Tyrosine residues in rabbit skeletal muscle creatine kinase were classified into two states: (1) 17 residues with a pK of 10.5, and (2) 2 residues with a pK of 11.8, whereas with beef cardiac creatine kinase, all 16 tyrosine residues ionized with a pK of 10.5. 3. 3. Tyrosine residues in the 165.000 dalton protein component were classified into three states: (1) 30 tyrosine residues with a pK of 10.5, (2) 15 tyrosine residues with a pK of 11.8 and (3) 9 non-ionizable tyrosine residues. 4. 4. When the 165,000 dalton protein component was treated with 4 M guanidine hydrochloride, all of the tyrosine residues became ionizable with a pK value of 10.0.

Evaluation of the Electrostatic Interaction Parameter on a Simulated Protein Titration Curve

Abstract Values for the electrostatic interaction parameter, ω, derived from the potentiometric titration curves of proteins may be erroneous unless both the number of groups ionizing with the same ionization constants are known and the titration curve is obtained with great precision and accuracy. The nature of the errors which can occur in the evaluation of u is demonstrated for calculated curves constructed without electrostatic interaction assumed. Since in a protein, the number of groups (and their pK's) titrating in a given pH region is unknown and the accuracy of protein titrations is limited, we suggest that the titration of a protein can be adequately described as the sum of a series of mass-law expressions.

Stability of Proteins Small Globular Proteins

The chapter discusses the stability of proteins and presents the results obtained on small compact globular proteins, which represent one single cooperative system. Protein is a cooperative system and behaves in an all-or-none fashion. Sharp changes in the properties of a protein do not mean anything in themselves because sequential multistep transitions exhibit the same sharp sigmoidal changes in the observed parameters. The problem of stability of native proteins is closely connected with the problem of protein denaturation, as stability can be judged only by breaking the native structure—that is, denaturing protein by various treatments. The pH of the solution is one of the most important factors determining the state of a protein. Potentiometric titration of protein revealed that smooth changes are connected with the titration of groups with a pK not very different from that of free amino acids, while the gross conformational changes associated with pH denaturation are accompanied by the unmasking of buried groups. The chapter also discusses the temperature-induced changes in protein, denaturational and predenaturational changes in protein, thermodynamics of protein unfolding, and thermodynamic properties of protein.

Intrinsic electric fields and proton diffusion in immobilized protein membranes. Effects of electrolytes and buffers

We present a theory for proton diffusion through an immobilized protein membrane perfused with an electrolyte and a buffer. Using a Nernst-Planck equation for each species and assuming local charge neutrality, we obtain two coupled nonlinear diffusion equations with new diffusion coefficients dependent on the concentration of all species, the diffusion constants or mobilities of the buffers and salts, the pH-derivative of the titration curves of the mobile buffer and the immobilized protein, and the derivative with respect to ionic strength of the protein titration curve. Transient time scales are locally pH-dependent because of protonation-deprotonation reactions with the fixed protein and are ionic strength-dependent because salts provide charge carriers to shield internal electric fields. Intrinsic electric fields arise proportional to the gradient of an "effective" charge concentration. The field may reverse locally if buffer concentrations are large (greater to or equal to 0.1 M) and if the diffusivity of the electrolyte species is sufficiently small. The "ideal" electrolyte case (where each species has the same diffusivity) reduces to a simple form. We apply these theoretical considerations to membranes composed of papain and bovine serum albumin (BSA) and show that intrinsic electric fields greatly enhance the mobility of protons when the ionic strength of the salts is smaller than 0.1 M. These results are consistent with experiments where pH changes are observed to depend strongly on buffer, salt, and proton concentrations in baths adjacent to the membranes.

Protein titration curves by combined isoelectric focusing-electrophoresis with hemoglobin mutants as models

By performing electrphoresis perpendicular to a stationary pH gradient generated by focused carrier ampholytes in a gel slab, a pictorial representation of a protein titration curve is obtained. By running a protein and its genetic mutants in parallel, the shape of the relative titration curve reveals which charged amino acid has been substituted. Within a given family of proteins, under a constant set of experimental conditions, the relative mobility at any given pH can be correlated with the number of protons lost or acquired by the protein.

Properties of the nitrogenase system from a photosynthetic bacterium, Rhodospirillum rubrum

Soluble nitrogenase from Rhodospirillum rubrum has been isolated and separated into its two components, the MoFe protein and the Fe protein. The MoFe protein has been purified to near homogeneity and has a molecular weight of 215 000. It contains two Mo, 25–30 Fe and 19–22 acid-labile sulphide and consists of four subunits, M w 56 000. The Fe protein has a molecular weight of 65 000. It contains approximately four Fe and four acidlabile sulphide and consists of two subunits, M w 31 500. The highest specific activities for the purified components are 920 and 1260 nmol ethylene produced per min per mg protein, respectively. The purified components require the membrane component for activity (Nordlund, S., Eriksson, U. and Baltscheffsky, H. (1977) Biochim. Biophys. Acta 462, 187–195). Titration of the MoFe protein with the Fe protein shows saturation and excess MoFe protein over Fe protein is inhibitory. Addition of Fe 2+ or Mn 2+ to the reaction mixture increases the activity apparently through interaction with the membrane component.

Isolation of Precursors of Cytochrome Oxidase from Neurospora crassa: Application of Subunit-Specific Antibodies and Protein A from Staphylococcus aureus

A novel immunological procedure has been applied for the isolation of precursors of cytochrome oxidase. It involves antibodies to individual subunits of the oxidase and protein A from Staphylococcus aureus linked to a Sepharose support. Unassembled (free) 'subunits' as well as a labile complex containing five polypeptide components of the oxidase were isolated from mitochondrial extracts by this technique. The procedure is superior to the previously used double-immuno-precipitation method, because of its quantitative nature, its sensitivity, rapidity and versatility. Thus, sequential titrations of precursor proteins by addition of various subunit-specific immuno-globulins to an individual extract become feasible. Furthermore, the technique is suitable for the isolation of precursors on a large scale. To avoid contamination of the polypeptide preparations with immunoglobulin, the antibodies were covalently coupled to protein A, which had been previously linked to Sepharose. A radioactive preparation of unassembled subunit 1 of cytochrome oxidase was isolated by such a modified support and its cyanogen-bromide-cleavage products were compared to those of subunit 1 obtained from the assembled enzyme.

Microbiological titration of proteins and of single amino acid content in biological materials without purification and hydrolysis

A method is described for the microbiological determination of the protein content of biological materials. This method can also be adopted to titrate the concentration of a single amino acid in the protein and has the following advantages: (1) titration can be done without purification and hydrolysis of proteins; (2) the titration graph is a straight line between 25 and 800 microgram/ml; (3) protein values agree with those obtained using the Kjeldhal method; and (4) each mutant requiring one amino acid may be used to titrate the concentration of a single amino acid of the protein. The leucine content of various kinds of flour was measured with this system.

Ionization behaviour of native apolipoproteins and of their complexes with lecithin. 1. Calorimetric and potentiometric titration of the native apoA-I protein and of the apoA-I protein-dimyristoyl lecithin complex.

The ionization behaviour of native apoA-I protein is compare to that of its complex with synthetic dimyristoyl lecithin in studies using calorimetric, potentiometric and spectrophotometric titration. In the presence of phospholipids, 10 out of 21 lysines together with 22 acidic residues are masked in the complex. All tyrosines remain accessible to titration below pH 13. The apparent ionization enthalpy of the 11 lysine residues is not affected by the presence of phospholipids. These data are consistent with discrete binding sites located in the apoprotein helical segments as suggested by the model of Segrest et al. [FEBS Lett. 38, 247-253 (1974)]. A tentative localisation of lysine, arginine, aspartic acid and glutamic acid residues directly involved in phospholipid binding is suggested, assuming that such helical regions are involved in apoprotein-phospholipid association.

On a new approximation in the theory of electrolyte solutions

We compare the results of a new approximation for the interionic radial distribution function developed previously by Olivares and McQuarrie to those from the nonlinear Poisson—Boltzmann equation for the highly-charged system of the spherical protein bearing a charge as 20 in an aqueous electrolyte solution. In particular, we use both approximations to predict the experimental results for protein titration, which are the experimental data to which the results of the Poisson—Boltzmann equation had been directed.

1H Nmr studies at 360 MHz of the methyl groups in native and chemically modified basic pancreatic trypsin inhibitor (BPTI).

In the 1H NMR spectra obtained at 360 MHz after digital resolution enhancement, the multiplet resonances of the methyl groups in the basic pancreatic trypsin inhibitor (BPTI) were resolved. With suitable double irradiation techniques the individual methyl resonances were assigned to the different types of aliphatic amino acid residues. Furthermore, from pH titration and comparison of the native protein with chemically modified BPTI, the resonance lines of Ala 16 in the active site and Ala 58 at the C-terminus were identified. Potential applications of the resolved methyl resonances as natural NMR probes for studies of the molecular conformation are discussed.

Bestimmung von Sulfhydryl und Disulfid-Gruppen in Proteinen mit Hilfe der amperometrischen Titration

The specifity of Ag+ ions for protein SH groups has been questioned frequently, even though the amperometric titration with AgNO3 is one of the most common methods for the determination of SH groups in proteins. This is due to the fact, that the formation of silver complexes in the titration of cysteine causes a consumption of AgNO3 which is too high. In order to find out if this may be true in the case of proteins, in the present work select proteins with a well known content of SH and SS groups have been titrated amperometrically in tris buffer pH 7.4 with 0.001 M AgNO3. The proteins used were hemoglobin, bovine serum albumin, ovalbumin, lysozyme, pepsin, myoglobin, and cytochrome c. The direct and the indirect titrations of (a) native, (b) denatured, and (c) NaBH4 reduced proteins showed, that the expected consumption of AgNO3 was in no case exceeded. Therefore under the conditions used AgNO3 may be considered as a specific reagent for protein SH groups. High SH values as a result of the amperometric titration of proteins with silver nitrate, which have been published occasionally, may be due to incorrect estimation of the end point of the titration. The reducibility of SS groups depends on the kind of protein. Lysozyme and pepsin were already completely reduced at 23 degrees C, whereas bovine serum albumin needed 60 degrees C. The direct titration method was useful only in some cases for the detection of all SH groups originally present in the proteins or formed by reduction with NaBH4. On the other hand the indirect titration method gave maximum values, because the slowly reacting SH groups of proteins are also allowed to react and the resulting titration curves may be evaluated correctly.

Studies on the assembly of a spherical plant virus: II. The mechanism of protein aggregation and virus swelling

Potentiometric and spectrophotometric titrations have been undertaken to understand the mechanisms of control of the pH-dependent swelling of cowpea chlorotic mottle virus and of the formation of capsid by the viral protein. The results show a striking difference in the proton release by the virion and by the protein. Both the swelling of the virus and the capsid formation are accompanied by an anomalous proton release which shows a well-defined hysteresis in the pH range where these phenomena take place. The swelling is not accompanied by any major change of the u.v. absorption, but only by a small decrease (2% at 260 nm). Magnesium ions, which have a marked effect on the swelling, have no effect on the protein titration, but abolish the hysteresis and suppress the small decrease in absorbance associated with the swelling. The data suggest that two carboxyl groups with a p K near neutrality are responsible for controlling the swelling, and also probably control the capsid assembly. The effect of magnesium ions is presumably on the nucleic acid configuration and (or) the RNA-protein salt links. The existence of hysteresis, both in the formation of capsids from protein and in the swelling of the virus, is an interesting feature, showing the existence of a pathway through metastable states.

Potentiometric titration of soluble and bound iron-sulfur proteins monitored by circular dichroism and electron paramagnetic resonance spectroscopy

Potentiometric titrations of iron-sulfur proteins monitored by changes in circular dichroism (CD) or electron-paramagnetic-resonance (EPR) spectrum are presented. Both monitoring techniques have the distinct advantage that interferences caused by the absorption changes of the redox mediators during a titration, which limit the use of the absorption spectrophotometry for monitoring, are either not monitored, or do not represent a serious interference. The CD tritration is illustrated with soluble spinach ferrodoxin and the EPR titration with bound iron-sulfur proteins of green plant photosystem I. The anacrobic titration apparatus and other experimental considerations are described in detail.

Approximative Berechnung der Pufferbase, einer Tittrationsgeraden f�r Proteine und der CO2-Dissoziationskurve des gehirngewebes

An analysis of the acid-base balance and the CO2-binding capacity of the brain is presented. It is based on a linear titration curve for the cerebral proteins, the mass action laws for the first dissociation of carbonic acid and the second dissociation of phosphoric acid, the condition of electrical neutrality and finally the experimental brain buffer line (pH=a log\(P_{Bco_2 } \)+b) based on the data of Kjallquistet al. (1969), the total phosphate ion and protein concentration of McIlwain and Bachelard (1971). The following values for the slope of the protein titration curve (d[P−]/dpH), an average isoelectric point of the proteins involved and the buffer base of whole brain were obtained: 37.18 meq/kg H2O·pH; 5.718; 77 meq/kg H2O. The CO2 dissociation curve derived from these data approximates well the experimental data of Kjallquistet al.

Coulometric titration of proteins with silver(I)

The sulfhydryl groups of some proteins were studied by a coulometric argentometric titration; disulfide groups in the presence of sulfite were titrated by the same technique. Direct procedures and a technique utilizing excess coulometrically generated silver(I) were used. The latter method enabled small samples (20–1000 μg of protein) to be titrated with a precision of less than 4%. The proteins were generally titrated in both the native state and the denatured state (8 M urea). Bovine serum albumin, α-chymotrypsin, glutathione reductase, human hemoglobin, insulin, β-lactoglobulin, and ovalbumin were examined and the results compared to those of previous workers.

Calorimetric titration of lysozyme.

A method is described for enthalpy titration of proteins, using a flow microcalorimeter in combination with a device for continuous pH measurements.The calorimetric titration of lysozyme between pH 1.5 and 12 has been carried out as well as the corresponding potentiometric titration. Enthalpy values for the titrated groups are estimated and discussed in comparison with reported data for free amino acids.

Interpretation of protein titration curves. Application to lysozyme.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTInterpretation of protein titration curves. Application to lysozymeCharles Tanford and Robert RoxbyCite this: Biochemistry 1972, 11, 11, 2192–2198Publication Date (Print):May 1, 1972Publication History Published online1 May 2002Published inissue 1 May 1972https://doi.org/10.1021/bi00761a029RIGHTS & PERMISSIONSArticle Views1887Altmetric-Citations407LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (829 KB) Get e-Alerts Get e-Alerts

Studies on protein sulfenyl iodide derivatives of β-lactoglobulin and bovine serum albumin

Whereas the treatment of β-lactoglobulin with I 3 + KI produced yellow solutions, as described by Cunningham and others, the treatment with aqueous solution of I 2 produced colorless ones. Aqueous I 2 produced protein sulfenyl derivatives with solutions of β-lactoglobulin or bovine serum albumin in a similar manner as the treatment of such solutions with 0.01 M I 3 − in the presence of 0.2 M KI did. Evidence is presented that the sulfenyl iodide group in protein derivatives is not a chromophore. A yellow colored solution was obtained after treatment of the colorless bovine serum albumin sulfenyl iodide solution with additional aqueous I 2 reagent. A similar yellow color was found spectrophotometrically in solutions of β-lactoglobulin sulfenyl iodide treated with additional I 2. Excess of the I 2 reagent produced blue colored protein-iodine complexes with a number of protein solutions. Spectral and chemical data on solutions of β-lactoglobulin and bovine serum albumin sulfenyl iodide derivatives treated with additional I 2 were compared with those of model iodide and periodide compounds. It is concluded that the yellow color appearing to varying intensity in iodimetric titrations of various thiol proteins with I 3 − or I 2 is due to the formation of protein derivatives with the character of a sulfenyl periodide.