Ribonuclease spectrophotometric titrations

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 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. 

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... 

Assay of Ribonuclease. Several kinds of assay have been found useful for studying ribonuclease (RNAase). A spectrophotometric assay is based on the empirical, unexplained observation that there is a decrease in optical density at 300 m/ during hydrolysis of RNA. RNA can be precipitated from solutions of its degradation products by uranyl acetate in trichloroacetic acid the soluble nucleotides can be measured spectro-photometrically or chemically. A manometric assay determines the formation of secondary acid groups through release of CO2 from a bicarbonate buffer acid groups have also been determined by titration. Recently cyclic nucleotides have been used as substrates in place of nucleic acid. ... 

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