Lysozyme abnormal carboxyl groups

Carboxyl groups absorb at about 1710 cm1, and carboxylates at about 1570 cm-1. Infrared difference spectra in D20 solutions have been used to measure the pA s of abnormal carboxyl groups in a-lactoglobulin (7.5) and lysozyme (2.0, 6.5). 

The titration curves of lysozyme in K and in G indicate that the various carboxyl groups (the three extra ones as well as some of the remaining nine groups) have different pK s. It is possible to determine the number of each kind of group (and the corresponding pK) by means of experiments in which the kinetics of the uptake or release of hydrogen ion is measured upon addition of G to lysozyme solutions. This determination is possible because the rates of normalization of the various abnormal carboxyl groups in G differ. The specific quantities obtained from the kinetic experiments are the number of protons taken up per molecule (Ar) and the pH dependence... 

This kinetic study indicates that only about three of the carboxyl groups in lysozyme which are titratable in KCl solution have abnormal pK s. The discrepancy in the number of groups between this kinetic study and the calculations of Tanford and Wagner (1954) and Beychok and Warner (1959) may arise because the kinetic method does not detect the abnormal carboxyl groups which normalize rapidly in G. 

Further information about the abnormal carboxyl groups in lysozyme has been obtained from studies of its ultraviolet difference spectrum. Unlike insulin (Laskowski et al., 1960a) and ribonuclease (Scheraga, 1957), where the difference spectrum is due to perturbations of the tyrosyl chromo-phore, there is no difference spectrum arising from perturbations of the tyrosyl groups of lysozyme by acid treatment. Instead, the acid treatment produces a difference spectrum in lysozyme which is due to perturbation of the tryptophan chromophore. 

The titration curve of egg white lysozyme is known to be abnormal in the regions of ionization of the carboxyl groups and of the tyrosyl groups (Tanford and Wagner, 1954). It therefore appeared likely that these groups might be involved in side-chain interactions which would make their pK s abnormal, and the following experiments were carried out to determine the nature of these interactions. 

The titration curve of native lysozyme in 0.15 molar KCl (designated K) is shown in Fig. 141. There are nine titratable carboxyl groups and the titration curve is abnormally flat, i.e., r does not increase rapidly enough with... 

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... 

In summary, these experiments illustrate the use of titration curves and ultraviolet difference spectra to obtain information about interactions which render some of the carboxyl groups of lysozyme abnormal. 

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