Protein titration

In molecular mechanics and molecular dynamics studies of proteins, assig-ment of standard, non-dynamical ionization states of protein titratable groups is a common practice. This assumption seems to be well justified because proton exchange times between protein and solution usually far exceed the time range of the MD simulations. We investigated to what extent the assumed protonation state of a protein influences its molecular dynamics trajectory, and how often our titration algorithm predicted ionization states identical to those imposed on the groups, when applied to a set of structures derived from a molecular dynamics trajectory [34]. As a model we took the bovine... 

Tanford, C., Kirkwood, J. G. Theory of protein titration curves. I. General equations for impenetrable spheres. J. Am. Chem. Soc. 79 (1957) 5333-5339. 6. Garrett, A. J. M., Poladian, L. Refined derivation, exact solutions, and singular limits of the Poisson-Boltzmann equation. Ann. Phys. 188 (1988) 386-435. Sharp, K. A., Honig, B. Electrostatic interactions in macromolecules. Theory and applications. Ann. Rev. Biophys. Chem. 19 (1990) 301-332. 

Tanford, C., Roxby, R. Interpretation of protein titration curves Application to lysozyme. Biochem. 11 (1972) 2192-2198. 

Tanford, C, Theory of Protein Titration Curves. 11. Calculations for Simple Models at Low Ionic Strength, Journal of the American Chemical Society 79, 5340, 1957. 

Tanford, C Roxy, Interpretation of Protein Titration Curves Application to Lysozyme, Biochemistry 11, 2192, 1972. 

Tanford C, Kirkwood JG (1957) Theory of protein titration curves. I. general equations for impenetrable spheres. J Am Chem Soc 79 5333—5339. 

Many amine bases and carboxylic acids in proteins titrate with anomalously high or low pA s (Table 5.4). The reasons are quite straightforward, and depend on the microenvironment. If a carboxyl group is in a region of relatively low polarity, its pKa will be raised, since the anionic form is destabilized. Alternatively, if the carboxylate ion forms a salt bridge with an ammonium ion, it will be stabi-... 

Cardamone and Puri (1992) stated that ANS binding and resultant Ka measured by a Scatchard plot or Kloz plot (and to a lesser extent quantum yield) may be used as a measure of the relative surface hydrophobicity of proteins. Titration of protein solutions with increasing concentrations of the fluorescent probe can provide information on both the number and the affinity of binding sites. This may be useful in determining whether the high value of fluorescence resulted from the presence of many binding sites of only moderate hydrophilic character, or from the existence of a high-affinity site with considerable hydrophilic character. 

The acid-base behavior of proteins can reveal some important properties with respect to both their composition (selectivity) and their concentration (sensitivity). The most direct way to exploit these acid-base properties is to make use of acid-base titration, Titrant should be added somehow and the resulting change in pH should be measured. Since the ion-sensitive field-effect transistor (ISFET) is suitable for fast (and local) pH detection, an ISFET can be used for protein titration if the protein to be detected can be immobilized in a membrane, deposited on top of the device. Advantages are the small amount of protein necessary for the characterization owing to the small membrane volume, and the relatively short time needed to perform a full titration. 

In addition to the methods described above, the dependence of a protein titration curve on the salt concentration can also be exploited by making use of the ion step method. The essential aspect of this approach is, as with the previously de-... 

We have presented four alternative methods for volumetric protein titration, based on a membrane-covered ISFET, in which proteins can be incubated. These methods are... 

The relation between structure and acidity of organic compounds has been the subject of much study. Those aspects which are of interest in connection with protein titration curves have been reviewed in definitive manner by Edsall and Wyman (1958) and by Edsall (1943), and the reader is referred to these reviews for a discussion of the theoretical and empirical principles which are involved. For the present purpose it is sufficient to extract the data which will lead to the expected pK values of the titratable groups of proteins, and this has been done in Table I. 

The assumption that protein molecules do not have unique interactions absent in smaller molecules is of course naive. It is in fact untrue. Special interactions occur and upset the expectations with which this section has been concerned. It is the occurrence of such deviations from the expected result which lend interest and importance to the study of protein titration curves. 

There is a simple way to avoid these problems. One can define a pH scale in a completely arbitrary manner relative to the emf of a suitable cell. One can then relate the pH on this scale to an arbitrarily defined activity of hydrogen ions, simply be setting pH = —log an+. The dissociation constants of model compounds can then be determined in terms of this arbitrary scale. This method has been used by Donovan et ai. (1959) for protein titrations in concentrated aqueous solutions of guanidine hydrochloride and of urea, and by Sage and Singer (1962) for titrations in ethylene glycol. 

Large sections of protein titration curves are often equally time-independent and reversible, as, for instance, the acid part of the titration curve of (3-lactoglobulin shown in Fig. 2. Any such section of the titration curve will again represent thermodynamic equilibrium and it may be subjected to thermodynamic analysis, as outlined in Sections VI and VII. 

A summary of information on the reversibility of protein titration curves,... 

The fact that a protein titration curve is reversible over a given range... 

Table V shows that the vast majority of the titratable groups of the smaller protein molecules have pK nt values which are quite close to the values predicted from the pK s of model compounds. This feature of protein titration curves has been well known for a long time, and is accepted as normal. It is however really an astonishing result, for it implies that most of the titratable groups of the smaller protein molecules are in as intimate contact with the solvent as similar groups on smaller molecules, and that they are able to accept or release hydrogen ions in this location without requiring any modification of the protein conformation in the vicinity of the titratable group. Since most of the proteins examined have been globular proteins, tightly folded so as to exclude solvent from most of the interior portions, the titratable groups must be nearly always at the surface.

The first detailed analysis of a protein titration curve, according to the semiempirical treatment used for most of the titration curves reviewed in this paper, also involves ovalbumin (Cannan el al., 1941). The first discovery of phenolic groups inaccessible to titration was again made with this protein (Crammer and Neuberger, 1943). 

Polar fishes, freezing resistance of, 195 12-Propanediol-water mixture dielectric constant of, 92 Protein fractionation at subzero temperatures, 77 -189 acid-base equilibria, 100-122 applications, 146-185 column chromatography, 140-141 density and viscosity changes, 82-85 dielectric constant variations, 85-99 general principles, 135-140 isoelectric focusing, 141-144 methods, 140-146 physicochemical data, 78-134 protein dissociation, 129-134 protein titration, 116-122 solubility of salts and solutes, 122-129... 

In most protein titration curves, including the two examples already given, only two discrete steps are readily discernible, and it is difficult to pick out even from these the numbers and dissociation constants of sets of... 

Fig. 6. (A) EPR spectra of isolated native FeS-A/FeS-B protein titrated to -495 and -599 mV, and measured at 10 K (left column) and 38 K (right column) (B) redox-titration plot of the EPR-signal intensity at g=1.91 measured at 10 K (open circles) and 38 K (solid dots). Figure source Oh-oka, Itoh, Saeki, Takahashi and Matsubara (1991) F/jF protein from spinach photosystem I complex Isolation in a native state and some properties of the iron-sulfur clusters. Plant Cell Physiol 32 14,15.

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