Protein from titration curves

The existence of these earlier reviews makes it possible for the present treatment to be limited in scope. It will be sufficient to touch only superficially on experimental techniques and on the theoretical derivation of equations The major objective will be, as the title of the paper implies, to show what one can learn from titration curves that is of general interest to protein chemistry. 

Next, it is worth while comparing the data from titration curves with those from electrophoresis. Here again the best investigations have been made on corpuscular proteins. A beautiful example is to be found in the work of Cannan, Kibrick and Palmer on the titration and that of Longsworth on the electrophoresis of ovalbumin. 

The charge (negative or positive net charge) of, e. g. a protein particle, is closely related to its electrophoretic mobility. Approximate information about the likely direction and magnitude of mobility of particles migrating in an electric field, can be obtained from titration results. There is, however, no quantitative agreement between the net charge as calculated from titration curves and from electrophoretic mobility. 

Ribonuclease is an enzyme with 124 amino acids. Its function is to cleave ribonucleic acid (RNA) into small fragments. A solution containing pure protein, with no other ions present except H+ and OH- derived from the protein and water, is said to be isoionic. From this point near pH 9.6 in the graph, the protein can be titrated with acid or base. Of the 124 amino acids, 16 can be protonated by acid and 20 can lose protons to added base. From the shape of the titration curve, it is possible to deduce the approximate pATa for each titratable group.1-2 This information provides insight into the environment of that amino acid in the protein. In ribonuclease, three tyrosine residues have "normal values of pATa(=10) (Table 10-1) and three others have pA a >12. The interpretation is that three tyrosine groups are accessible to OH, and three are buried inside the protein where they cannot be easily titrated. The solid line in the illustration is calculated from pA"a values for all titratable groups. 

Theoretical titration curves for enzymes can be calculated from known crystal structures and first principles of electrostatics. Key amino acids at the active site have significantly perturbed pK values and unusual regions in which they are partially protonated over a wide pH region.3 In principle, such titration calculations can identify the active site of a protein whose structure is known, but whose function is not. 

Electrostatic theory has also been used successfully to interpret titration curves of proteins in which the net negative or positive charge distributed over the surface of the protein varies continuously from high pH to low as more protons are added.19... 

An acid-base conjugate pair can act as a buffer, resisting changes in pH. From a titration curve of an acid the inflexion point indicates the pK value. The buffering capacity of the acid-base pair is the ptC 1 pH unit. In biological fluids the phosphate and carbonate ions act as buffers. Amino acids, proteins, nucleic acids and lipids also have some buffering capacity. In the laboratory other compounds, such as TRIS, are used to buffer solutions at the appropriate pH. 

Workers in the laboratory of R. C. Warner (133, 139) have examined many different aspects of the charge relationships of chicken ovotrans-ferrins. Fig. 3 is a reproduction of titration curves of ovotransferrin at different temperatures and different ionic strengths. Other data from the exacting experiments of these investigators should be consulted by those interested in the general physical chemistry of proteins. The data particularly pertinent to the chelation of metal ions will be discussed in subsequent sections. 

The number of electrons transferred per mole of adrenodoxin (n-value), determined from the slope of the potentiometric titration curve, is 2. This result is further supported by anaerobic titration of the protein with NADPH in the presence of adrenodoxin reductase (30). The result shows that one mole of NADPH is required to reduce one mole of adrenodoxin. Therefore, it is concluded that adrenodoxin transfers two electrons per mole of the protein. Obviously, this conclusion is incompatible with the EPR data which favor the interpretation that one of the two irons is reduced. This discrepancy can not presently be understood in detail. 

From this point of view, titration curves do not represent just another way of physically characterizing a protein molecule. More than most other physicochemical methods which are in common use, titration studies tend to emphasize individual differences among proteins, and this is reflected in the organization of this paper. There is a large section, entitled Results for Individual Proteins, which contains the many features of... 

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. 

The procedure described in the preceding paragraph will of course measure the number of hydrogen ions bound to or dissociated from all substances which are present in the solution under study. The accuracy of an experimental electrometric titration curve depends to a considerable degree on the absence of buffers, carbon dioxide, and any other substance, other than the protein of interest, which is capable of acting as an acid or base. 

Titration curves may sometimes depend on the time which has elapsed, between addition of acid or base and the measurement of pH. (This is true, for instance, of the alkaline part of the curve shown in I dg. 2.) By the same token, the titration curve obtained by addition of successive increments of acid or base to the same protein solution will sometimes differ from the curve obtained by addition of successively larger increments of acid or base, each to a fresh aliquot of the initial protein solution. 

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