Ribonuclease titration

The presented algorithm was applied to 4 proteins (lysozyme, ribonuclease A, ovomucid and bovine pancreatic trypsin inhibitor) containing 51 titratable residues with experimentally known pKaS [32, 33]. Fig. 2 shows the correlation between the experimental and calculated pKaS. The linear correlation coefficient is r = 0.952 the slope of the line is A = 1.028 and the intercept is B = -0.104. This shows that the overall agreement between the experimental and predicted pKaS is good. 

Acid-base titration of the enzyme ribonuclease. The isoionic point is the pH of the pure protein with no ions present except H+ and OH. The isoelectric point is the pH at which the average charge on the protein is 0. [C. I Tanford and J. D. Hauenstein, Hydrogen Ion Equilibria of Ribonuclease." J. Am. Chem. Soc. 1956, 78.5287.]... 

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. 

In the titration of ribonuclease at the beginning of this chapter, there is a continuous change in pH, with no clear breaks. The reason is that 29 groups are titrated in the pH interval shown. The 29 end points are so close together that a nearly uniform rise results. The curve can be analyzed to find the many pKa values, but this analysis requires a computer and, even then, the individual pKa values will not be determined very precisely. 

Ribonuclease contains no tryptophan. The absorption near 280 nm is almost entirely resulting from the 6 tyrosine residues. The ionization of tyrosine produces a marked shift to longer wavelengths in the absorption spectrum. The ionization can be monitored near 295 nm. Shugar (293) was the first to point out the abnormal behavior of 3 of the tyrosine residues on alkaline titration. Three titrate normally with apparent pK values near 10, but three do not titrate until much more alkaline pH values have been reached and irreversible alkaline de-naturation has set in. Some typical spectra and difference spectra are... 

The tyrosines of bovine pancreatic ribonuclease (RNase) appear to be a case in point. Three of the total of six tyrosine residues per RNase molecule titrate reversibly with a normal pK of about 10, but the other three titrate only at much higher pH and then irreversibly (Shugar, 1952 Tanford et al., 1955a). These results suggest that the RNase molecule has to undergo a profound structural rearrangement before the three anomalous tyrosines become accessible to titration. Furthermore, the absorption of RNase due to tyrosine residues at about 280 m/i exhibits a hyperchromic effect presumably as a result of the special environment of the three anomalous tyrosines. 

Sage and Singer (1958, 1962) showed that ribonuclease could not only be recovered from neutral ethylene glycol into aqueous solution with essentially full retention of enzymatic activity, but that this was so even after all six of its tyrosine residues had been converted to the phenoxide ion form in ethylene glycol. This is in contrast to the situation in water solutions of this protein, in which the titration of more than three of the six tyrosines results in an essentially instantaneous irreversible loss of enzymatic activity (Sela and Anfinsen, 1957). This suggests the interesting possibility that the irreversible transition that occurs in aqueous solutions... 

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). 

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.]...

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. 

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). 

The titration curve of ribonuclease (Tanford and Hauenstein, 1956b) is reversible between its acid end point and the onset of alkaline denaturation. All titratable groups, which are expected to be present on the basis of amino acid analysis, are found to be titrated in the expected parts of the titration curve, with the exception of the abnormal phenolic groups mentioned above. The amino and imidazole groups appear to have normal pK s, and the neutral and alkaline regions in which they occur are compatible with the same values of w as are required to fit the titration curves of the three normal phenolic 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. 

Ribonuclease All 6 Tyr s titrate as one class in 8 M urea Blumenfeld and Levy (1958)... 

Ribonuclease in urea-guanidinium chlo- All Tyr s titrate as one group electrostatic factor high Cha and Scheraga (1960)... 

The use of buffer solutions is usually advisable for accurate control of the pH of solutions used in spectrophotometric titrations, since the solutions will usually be so dilute (because of the relatively high protein or peptide absorptivity) as to offer little self-buffering. The usual buffers for the pH range from 9 to 13, i.e., borate, glycinate, phosphate, lysine, -aminocaproic acid, etc., are generally transparent through the 2950 A phenolate band. Buffer systems composed of piperidine (pK, 11) and its hydrochloride have been used to avoid the use of multivalent anions in the spectrophotometric titration of ribonuclease (Klee and Richards, 1957), but no other advantage is apparent for this system. 

Another important feature of Fig. 19 is that it shows a twofold increase in the solvent-accessibility of the tyrosyl groups of oxidized ribonuclease (and acid-denatured) compared to the native protein. This result fits nicely with the findings of Shugar (1952) and of Tanford et al. (1955), who showed by spectrophotometric titration that only three out of a total of six tyrosyl groups of ribonuclease can be titrated reversibly. It appears... 

Figure 13 shows these changes in extinction for the NBS titration of tryptophan in bovine serum albumin (Ramachandran and Witkop, 1959), which is dissolved in 10.0 M urea solution in order to make the tryptophan units accessible. Another convenient way of picturing the changes in extinction is shown in Fig. 14 (Peters, 1959). Here one recognizes at a glance that ribonuclease contains no tryptophan. Based on a value of 2.8 X 10 for the amplitude in drop of molar absorption of free tryptophan it was concluded that human serum albumin (HSA) contains one, bovine serum albumin (BSA) two, and ovalbumin probably four rather than three tryptophan units.

An exchangeable proton of ribonuclease A titrates with a pK of 5.8 and has been assigned [39] to the NH of the active-site histidine-119. A low field resonance can be observed for chymotrypsin in H2O [40] and its pH dependence (15 to 18 ppm, pKa 7.2) and response to chemical modification suggests that this is the hydrogen-bonded proton between His-57 and Asp-102 at the active site. 

Thus, as with ribonuclease hydrolysis, chemical hydrolysis of the polymer causes fission of a link involving a secondary acidic group of a phosphoryl radical it is not yet known (a) whether the products are identical, and (b) whether the same phospho groups are split in each type of depolymerization. Accepting Gulland s titration results, possible formulas for the tetranucleotide (in which polymerization takes place at A or B) are as follows. 

Nozaki, Y., and C. Tanford Proteins as random coils. II Hydrogen ion titration curve of Ribonuclease in 6 M guanidine-hydrochloride. J. Am. Chem. Soc. 89, 742 (1967). 

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