Tyrosine spectrophotometric titration

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. 

A solvent which has been foimd to be of great interest in connection with protein conformation studies is ethylene glycol. Sage and Singer (1958, 1962) have investigated in some detail the properties of RNase in pure ethylene glycol, containing added neutral electrolyte. They examined the ultraviolet absorption spectrum, the ionization behavior of the tyrosine residues by spectrophotometric titration experiments, and the optical rotatory dispersion of the system. 

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 difference absorption spectra of hen and turkey lysozymes in the alkaline pH region had three maxima at around 245, 292, and 300 nm and had no isosbestic points.The ratio of the extinction difference at 245 nm to that at 295 nm changed with pH. These spectral features are quite different from those observed when only L-tyrosyl residues are ionized, and it was impossible to determine precisely the pK values of the L-tyrosyl residues in lysozyme by spectrophotometric titration. A time-dependent spectral change was observed above about pH 12. This is not due to exposure of a buried L-tyrosyl residue on alkali denaturation. The disulphide bonds and the peptide bonds in the lysozyme molecule were cleaved by alkali above about pH 11. The intrinsic pK value of L-tyrosine-23 of hen lysozyme was determined to be 10.24 (apparent pK 9.8) at 0.1 ionic strength and 25 C from the c.d. titration data. Comparison of the c.d. 

The spectrophotometric technique exploits the fact that when it binds to transferrin, AF+ replaces hydroxyl protons from two tyrosines, thus causing a change in the UV region of the spectrum (Fig. 10). Titration of the spectral change as a function of [AP+] at constant transferrin concentration allows the binding stoichiometry and stability constant to be measured. This approach has been used to study the interaction of a large number of metals [e.g., Nd +, Sm , Zn +, and Ga 61, 62,136)] with transferrin, including AP+ (33, 43, 136, 138) some of these metals would otherwise be spectroscopically silent. Such... 

Kasper and Deutsch 124) have titrated the approximately 65 tyrosine residues spectrophotometrically. The titration between pH 9 and 12 was not reversible for either native or apo-cemloplcismin but the magnitude of the spectral changes and the degree of hysteresis observed on back titration from pH 13 were substantially the same with native apo forms. No indication was obtained that t u osine and residues were either iuvolved in the binding of Cu or participated in any unique structural feature of the enzyme. 

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