Phenolic groups spectrophotometric titration

An interesting example of difference counting is provided in the conal-bumin study. Conalbumin can bind two iron atoms very tightly and it had been concluded earlier (Warner and Weber, 1953) that each iron atom might be bound to three phenolic groups, which would remain ionized at all pH s where the iron complex is stable. This earlier conclusion was firmly established by spectrophotometric titration of the iron complex. Only five phenolic groups were titrated between pH 8 and 12, compared to eleven in native iron-free conalbumin. The result shows, incidentally. 

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. 

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.

Figure 13 shows the data for the three phenolic groups of ribonuclease which ionize reversibly (Tanford etal., 1955a), based on spectrophotometric titration curves such as Fig. 11. A straight-line plot is obtained, in agreement with Eq. (14). The values of w are 0.112, 0.093, and 0.061, respectively, at ionic strengths 0.01, 0.03, and 0.15. (The salt used to produce the ionic strength was KCl, and there is evidence that neither K" nor CF is bound to an appreciable extent. The use of Zn as abscissa is therefore presumably acceptable.) Comparison with the calculated values of Table III shows that the experimental values are lower than predicted by about 20%. Such a deviation must be considered almost within the error of calculation. [If the radius of the hydrodynamically equivalent sphere (19 A) had been used as the basis of calculation, the calculated values of w would have become 0.119, 0.096, and 0.066, respectively.]...

Titration curves for bovine a-chymotrypsinogen have been determined under a variety of conditions by Wilcox (1961). The results are summarized in Table IX. The spectrophotometric titration of the phenolic groups is shown in Fig. 4. 

A spectrophotometric titration of the phenolic groups of myosin and its subunits has been reported by Stracher (1960). The data resemble those shown for ribonuclease in Fig. 11. About two-thirds of the tyrosine residues are titrated normally, and about one-third appear inaccessible in native myosin. An interesting feature is that 6 M urea has no effect at all on the titration curve. 

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. 

Glazer and Smith (1961) have carried out a spectrophotometric titration of the phenolic groups of papain. Of the seventeen phenolic groups known to be present, eleven to twelve ionize normally (pKint = 9.8). The remainder ionize only upon denaturation, which takes place only slowly in the range of pH 12 to 13. 

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 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. 

Since the three extra carboxyl groups appear to be interacting with lysyl rather than with phenolic groups, the abnormally high pK a of the phenolic groups remain to be explained. While the nature of the interactions which make the phenolic groups of lysozyme abnormal is as yet unknown, it is possible to disrupt these interactions with GU at 25 C. Analysis of the spectrophotometric titration curve of Fig. 149 indicates that the... 

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).

---