Lysozyme titrations curves

Tanford, C., Roxby, R. Interpretation of protein titration curves Application to lysozyme. Biochem. 11 (1972) 2192-2198. 

Tanford, C Roxy, Interpretation of Protein Titration Curves Application to Lysozyme, Biochemistry 11, 2192, 1972. 

Figure 3-2 Acid-base titration curve for hen lysozyme at 0.1 ionic strength and 25°C. O, initial titration from the pH attained after dialysis , hack titration after exposure to pH 1.8 A, hack titration after exposure to pH 11.1. The solid curve was constructed on the basis of "intrinsic" pKa values based on NMR data. From Kuramitsu and Hamaguchi5...

As expected, the end point in the titration curve shifts to higher values for longer incubation times, indicating a larger amount of lysozyme entering the membrane. The time needed to pass pH 7 as a function of the lysozyme concentration at fixed incubation time is shown in Fig. 11. In Fig. 11, the relation between this time and the lysozyme concentration is shown for two different values of the actuator current. The higher this current, the more titrant per second is generated, and consequently the shorter the time it takes to pass pH 7. 

FIG. 10 Typical titration curves measured with a coulometric sensor-actuator device at 5 nA generating current before and after the mentioned number of minutes of incubation in 3- I0-5 M lysozyme. 

FIG. 11 The measured time needed to cross pH = 7 at the titration curve, /ph7. minus rpn7 of the bare membrane as a function of the lysozyme concentration after I h incubation. 

Titration studies of lysozyme have revealed two unique features, both occurring in the carboxyl region of the titration curve. The pertinent data are shown in Table XIV. It is seen (o) that the count of carboxyl groups varies widely from one preparation of lysozyme to another, and (5) that three extra carboxyl groups appear in denaturing solvents such as 8 Af guanidine hydrochloride. 

Lysozyme Tyr pK high, titration curve flat Tanford and Wagner (1954)... 

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... 

Figure 4. Titration curves for hen egg white lysozyme and thioredoxin disulfide radical. Red lysozyme blue ...

Phase boundaries were also developed for p-lactoglobulin, chicken egg albumin, lysozyme, ribonuclease, and trypsin, all at r=100, a weight ratio at which polymer saturation appears to take place (see Discussion section). For each protein, pHcritical was converted to net negative surface charge (Zpr) per unit protein surface area (A), using potentiometric titration curves (26-31) and hydrodynamic radii (32) found in literature. Plots of surface charge density (Zpr/A) vs. I are shown in Figure 3. 

Interpretation of Protein Titration Curves. Application to Lysozyme. S. J. Shire,... 

Kuramitsu, S., Hamaguchi, K. Analysis of the add-base titration curve of hen lysozyme. J. Biochem. (Tokyo) 1980,87,1215-9. 

Fig. 4. Effect of pH on the reactivity of histidine-15 of hen egg-white lysozyme. The solid line shows a theoretical titration curve with a pKa of 6.4 and a reactivity of 4.25.

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... 

Fig. 141. Titration curves of lysozyme in 8 M G (approximately 1.5 hours after solution of the protein) and in 0.15 M KCl (K), at 25°C. The values of r shown as ordinate have been chosen to correspond to the titration in G. The curve K has been moved vertically to coincide with the other at the higher pH values (Donovan el al.,...

Fig. 144. A plot of the titration curve of lysozyme in GU, according to Eq. (VII-12). The a-carboxyl group has been included and probably causes some of the discrepancy at high Z. The maximum value of Z was assumed to be 20 and no chloride binding was assumed. The value of w, calculated from the slope of the line, is 0.052 (Donovan ei al., 1960).

Fig. 148. Titration curves of the guanidinated lysozyme at 25 C. (Donovan efaf.. 1960).

The titration curve of the deaminated derivative in KCl (Fig. 147) shows approximately five more titratable acid groups than the native lysozyme but there is no increase in the number of these groups in G. Since nitrous acid has many side reactions, the large number of acid groups in this derivative is not too surprising. 

Although the titration curve of the guanidinated derivative in KCl (Fig. 148) does not show the three extra carboxyl groups, these groups appear when the titration is carried out in G. The titration curve of this derivative in KCl is almost identical, on, the acid side, with the KCl titration curve of the G-treated lysozyme, prepared by allowing native lysozyme to stand a few hours in 8 molar G and then removing the G by dialysis (see curve D of Fig. 143). These titration curves presumably differ from the titration cur eof the native material because of the different manner in which the molecule has refolded, after it was unfolded either by G or the conditions under which this derivative was prepared. 

The question then arises as to the reasons why the three carboxyl groups are masked in the native protein. If these three carboxylate ions are able to accept protons in KCl solution, they must do so below pH 2, since no indication of this is observed in the titration curve of lysozyme. If the assumptions made in Chapter II are correct, then either hydrogen bonding fails to explain this extreme lowering of the pK of these carboxyl groups, or rather unusual, and o priori unlikely, situations of cooperative hydrogen bonding exist in the lysozyme molecule. 

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... 

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