Proteins acid titration curves

Interpretation of Acid Titration Curves for Proteins in the Presence and Absence of Ligand Binding Potential of Wyman References... 

An acid titration curve can be represented by either one of these equations (14) This second form is useful in thinking about the titration curve of a protein that binds a ligand. The pKs of these independent groups can be divided into two classes, those that are not affected by the binding of the ligand and those that are. 

The theoretical description of the titration curves of proteins and polyprotic acids has been studied since the pioneering work of Linderstrom-Lang [2] and Tan-ford [3]. In the following I will introduce the theory presently used in the calculation and analysis of protein residue titration curves. 

Poly electrolytes. These are molecules with a large number of ionizing groups which may have the same charge, as in polyacrylic acid, or both charges may be present, as in proteins. The titration curves of poly electrolytes have a rather indefinite ( smeared-out ) appearance. Contrary to... 

With multiple ionizable groups, such as in amino acids and proteins, each group titrates separately according to its pKa. The titration curves shown in Fig. 23-5 are for the amino acids glycine, histidine, and glutamate. 

B. Although titration curves for proteins are complex because of their multiple acidic and basic groups, their behavior can be illustrated by titration of a simple amino acid such as alanine (Figure 2-1). 

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. 

Make a table of characteristic pKa values for acidic and basic groups in proteins. Which of these groups contribute most significantly to the titration curves of proteins ... 

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. 

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. 

Conclusion. If protein molecules exhibit no interactions that are not also present on smaller molecules, then the pK values of their titratable groups would be expected to be roughly those of Table I. Electrostatic forces may move them up or down by as much as 1.5 pK units, but relative values will be unaffected thereby. The pK changes during the course of titration, so that the titration curve for any one group will be broader than it would be for a monobasic acid. 

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. 

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. 

Fig. 8. Hypothetical titration curves illustrating time-dependence and irreversibility. Curve 1 is an apparently time-independent curve, obtained by continuous titration, waiting several minutes for each successive pH reading. Curve 2 is the reverse titration curve, beginning at the acid end point. Curve 3 is the forward titration curve obtained by flow methods, each pH being measured on a freshly mixed solution within seconds of mixing. Curves 4 and 5 are obtained from freshly mixed solutions with longer time intervals between mixing and measurement. Curve 6 is the titration curve which one might speculatively draw to represent instantaneous titration of the native protein.

Fetuin is a glycoprotein which has titratable groups associated with its carbohydrate moiety (sialic acid) in addition to those present on the protein. Spiro (1960) has determined the titration curve of the native protein, as well as that of a preparation from which sialic acid had been removed. The group count differed only in the number of groups assignable to sialic acid. In particular, the value of S was the same for both proteins. It is clear from these results that the combination of sialic acid with the protein does not involve any of the basic groups of the protein. 

A titration curve for sperm whale myoglobin has been reported by Bres-low and Gurd (1962). The most striking feature is that it exhibits a time-dependent acid denaturation, which resembles that observed for the similar protein hemoglobin. To elucidate the physical nature of this reaction, emphasis was placed on the back titration to neutral pH of denatured protein. As in the case of hemoglobin (mentioned earlier), there are two major differences between the titration curves of native and denatured myoglobin, as shown by the data of Table XV. 

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