Phenolic groups protein, titration

Another example is provided by the titration of the phenolic groups of ribonuclease (Tanford et al., 1955a), in 0.15 M KCl. At 25°C the data strongly suggest that only three out of six phenolic groups are titrated in the native protein. At 6°C this conclusion becomes unequivocal (Fig. 11). 

It is seen that all four phenolic groups are titrated together in 6.4 M urea, whereas only two are titrated in the native protein. Even these two have sufficiently different pK s so that the titration of one is essentially complete before that of the second has begun. 

Fig. 4. Spectrophotometric titration of the phenolic groups of a-chymotrypsino gen (Wilcox, 1961). The native curve was obtained at 25°C in 0.1 M KCl. The arrows indicate time-dependent data. The upper curve was obtained at 25°C in 6.4 M urea, containing 0.1 M KCl. Under these conditions the protein is denatured. The total number of groups titrated is 4, a change in absorbance of about 2000 at 295 m/t corresponding to titration of a single phenolic group.

More phenolic and amino groups are titrated slowly above pH 12 as the protein becomes denatured. 

Havsteen and Hess (1962) have studied the titration of the phenolic groups of a-chymotrypsin and of its diisopropylphosphoryl (DIP) derivative. The result is similar to that observed with chymotrypsinogen (Fig. 4) in that only two of the four groups are available for titration in the native protein, as well as in the DIP derivative. The data do not have sufficient precision to determine whether the two titratable groups have different pK s, as they do in chymotrypsinogen, but the wide spread of the titration curve suggests that they do. 

Vodrazka and Cejka (1961) titrated the phenolic groups spectrophoto-metrically, and found no evidence for buried groups. Hermans (1962), has reported from a similar study that four groups per molecule (one per polypeptide chain) are not titratable in the native protein. Hermans study employed absorption at 245 m/i instead of the customary wavelength of 295 m/i. 

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). 

Within the limits of error of amino acid analyses available at the time, the count of groups obtained by Cannan et al. agreed with expectation, except in so far as the alkaline part of the curve was concerned. The number of groups titrated here is essentially the same as the number of amino groups, rather than the sum of amino and phenolic groups. This result is in accord with the later spectrophotometric titration of phenohc groups essentially all of these groups are inaccessible to titration in the native protein. 

Spectrophotometric titration of the phenolic groups led to the conclusion that all of the fifty-eight phenolic groups present on each paramyosin molecule were titrated in the guanidine-urea mixture, but that only forty-nine were titrated in the native state in 0.3 M KCl. (All of these had an essentially normal pKi t of 9.6.) This conclusion however was based entirely on the fact that the total change in absorbance at 295 m/i, between neutral pH and pH 14, is about 15 % less in the native protein than in the denatured state. If the change in absorbance per group titrated were to differ in the two solvents, then the conclusion reached would have to be revised. 

The best-known feature of the titration of ribonuclease is the fact that only three of the six phenolic groups of this protein can be titrated while the protein is in its native state, as was first reported by Shugar (1952). 

These three groups have an essentially normal intrinsic pK and their titration curve yields essentially normal values for the interaction parameter w (Tanford et al., 1955a). The three phenolic groups which are not available for titration in the native protein are titrated at 25°C near pH 13, where the protein becomes denatured. At 6°C, where the rate of denaturation is slower, a pH of 14 can be reached with only partial titration of these groups. 

The study by Martin et al. is of interest not only for the rationalization of the electrometric and spectrophotometric measurements in terms of the microconstants, but also because the spectrophotometric titration of tyrosine relates so closely to similar studies in proteins. In particular, the multiple H+-equilibria of tjnrosine result from the close juxtaposition of amino and phenolic groups in the same molecule under these circumstances the ionizations are mutually interacting. We suggest that some of the anomahes seen in t3Tosyl ionization in proteins may arise in a similar fashion, but in terms of magnitude, this mechanism clearly cannot account for such anomalous tjn-osyl groups as those seen in ribonuclease or ovalbumin. 

Fig. 149. Spectrophotometric titration curves of the three phenolic groups in lysozyme at 25°C. The points are average values of the data obtained at three wavelengths 290, 295, and 300 m/ - Dashed curve the ionization of the phenolic groups after the protein was heated in 9 M urea at 60° for 24 hours. At pH 13.0, a value of 1.0 was assumed for the degree of ionization a. This curve is similar to that reported by Tanford and Wagner (1954) for the ionization of the phenolic groups in KCl solution, and to curves obtained in urea without heating (not shown). Solid curve GU at 25°C., the points on this curve having been determined after the protein had been in solution for about 2 hours. This sample was not heated (Donovan el al., 1960).

Iodination of proteins has produced measurable changes in the titration curves. In zein (280), insulin (125), and pepsin (6) the region of the titration curves usually assigned to the phenolic group of tyrosine (pK=10) is displaced in iodinated proteins in the direction of increased acidity by nearly 2 pH units. This is apparently not true for iodinated globin (299). 

With several other proteins, such as bovine serum albiunin (Tanford and Roberts, 1952), lysozyme (Tanford and Wagner, 1954), and/3-lacto-globulin (Tanford and Swanson, 1957), pK shifts of the phenolic OH groups of tyrosine residues are observed, but these are of a qualitatively different nature. Thus, the tyrosines of any one of these proteins cannot be readily differentiated into a normal and an abnormal variety, since the spectrophotometric titration data for these proteins are reversible and fall on single smooth curves, in contrast to the situation with RNase. On the other hand, the tyrosine residues of ovalbumin show comparable behavior to the three abnormal tyrosine groups of RNase (Crammer and Neuberger, 1943). About 2 of the total of 9 tyrosine residues appear to titrate normally, but the remainder are not titrated up to pH 12. At pH 13, these anomalous tyrosines become titratable, and this is accompanied by the irreversible denaturation of the ovalbumin molecule. 

Therefore, simple quantitative analysis based on UV measurements is possible for many proteins, especially those containing aromatic chromophores. Analysis protocols may exploit other physical measurements, such as the titration of the phenolic hydroxy group to determine the number of tyrosine residues present in a peptide or protein, by following changes in UV spectra of a protein as a function of pH. 

---