Protein intrinsic dissociation constants

In some cases the quantities p(/ Ci t), and m yielded by analysis of titration curves may furnish special clues to the structure of particular proteins. For example, if ordinarily dissociable groups participate in formation of intramolecular bonds abnormal values for their intrinsic dissociation constants may result, or their number may even appear to be smaller than that found by amino acid assay. Examples will be furnished in the later sections on stoichiometry and on unreactive prototropic groups. 

As stated earlier, we hope to determine complex stoichiometry through the use of the Hummel-Dreyer technique. A calculation of stoichiometry at the point of polymer saturation will provide the average number of binding sites per polymer molecule, from which an intrinsic dissociation constant may be estimated. Currently, calculations of complexation stoichiometries have been hampered by a chromatographic overlap of the protein peak with that of the complex, and also by the extremely high polydispersity of the PDMDAAC samples. 

Metalloenzyme is represented by E(Mg2 ), C is chelator, E(Mg2 C) represents chelator reversibly bound to the metalloenzyme with dissociation constant KD and with characteristic enhancement of the protein fluorescence, and k is the first-order rate constant for irreversible formation of inactive protein P and the cation-chelator complex, Mg2+C. Values of both Kd and k may vary according to the nature of the chelator, the nature of the metal ion (if other cations can replace Mg2 ), and the intrinsic stability of the cation-chelator complex, as well as pH, temperature, and protein concentration. 

There are two kinds of properties that characterize the parts (i) intrinsic properties, which are determined by the part itself, such as mass, or the amino acid sequence of a protein, and (ii) relational properties, which are determined not only by individual parts but also by one or more other parts, such as dissociation constants. In complex biochemical systems, aggregative system properties, such as the mass of a bacterium, are a function of only the intrinsic properties of the parts. However, the flow through a biochemical pathway is a nonlinear function of the concentrations of its constituent enzymes. Therefore, the flow is not an aggregative property. 

An alternative approach to binding is based on the Scatchard equation [96]. If a protein has n independent and identical binding sites with intrinsic binding constants K and a fraction 0 of these are occupied at a given surfactant concentration [S], then a simple kinetic argument, in which the rate of binding is proportional to [S] times the fraction of vacant sites (1-0) and is equated to the rate of dissociation from the occupied sites proportional to 0, gives... 

For deductions to be made as to the state of the aromatic amino acid residues in proteins, the conditions under which the amino acids and the proteins are examined must be similar. Thin-film data on crystalline material are scarcely comparable with those obtained from the study of the same compounds when combined in macromolecules in solution, although they may have intrinsic interest of their own. Similarly, caution must be observed in comparing data obtained at low temperatures with those at room temperature where there is a possibility that ionic dissociation is involved. The meaning of pH at low temperatures in solid media and the values of the relevent dissociation constants are scarcely possible of definition. Keilin and Hartree (1949) have demonstrated the decolorization of certain dyes and indicators on cooling, which they ascribe to decrease of ionization. For instance, a red phenol-phthalein solution at pH 10.0 becomes colorless on cooling to — 15°C. 

To evaluate binding affinities by means of fluorescence titrations, the protein concentration m the system should not be substantially greater than the dissociation constant. We have found that under our expenmental conditions a protein concentration of 0.05 iM was compatible with a signal of intrinsic protein fluorescence high... 

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