Lysozyme 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 useful test for spectrophotometric titrations is to compare the apparent tyrosyl ionization from absorptivity versus pH measurements at several wavelengths. A good illustration is found in Tanford and Wagner s (1954) study on lysozyme. Their measurements at 2880, 2900, and 2950 A resulted in nonidentical titration curves. From these results, they concluded that .. . the observed changes in light absorption are not a true... 

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. 

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).

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