Lysozyme titration

The presented algorithm was applied to 4 proteins (lysozyme, ribonuclease A, ovomucid and bovine pancreatic trypsin inhibitor) containing 51 titratable residues with experimentally known pKaS [32, 33]. Fig. 2 shows the correlation between the experimental and calculated pKaS. The linear correlation coefficient is r = 0.952 the slope of the line is A = 1.028 and the intercept is B = -0.104. This shows that the overall agreement between the experimental and predicted pKaS is good. 

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. 

Machuqueiro M, Baptista AM (2007). Stochastic titration study of hen egg lysozyme. Proteins 72 289-298. 

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...

Simulation Results. A onc-dimensional simulation model based on the Nernst-Planck and Poisson equations [14, in which all the acid-base reactions occurring in the membrane are taken into account, has been used to give a qualitative description of the pH step titration process. In these simulations, a pH step is applied outside a 2 mm thick stagnant layer, which is assumed to be present in front of an 8 mm thick membrane. Diffusion coefficients in the membrane are assumed to be 4/10 of those in water (this value is based on experience with ion step experiments). Lysozyme, used as a model protein, is assumed to contain 11 carboxylic groups (pKa = 4.4), 2 imidazole groups (pKa = 6.0), and 9 amino groups (pKa = 10.4) per molecule. Concern... 

As an example, the ISFET response to a pH step from 4 to 10 applied outside an 8 mm thick membrane containing lysozyme in equilibrium with a l M KNCh electrolyte solution is simulated. It should be noted that not only mobile protons react with hydroxyl ions but also protons that dissociate from acidic and basic lysozyme groups. These dissociation reactions strongly delay the titration process. 

These results show that without any optimization of membrane composition, measurement of lysozyme in concentrations ranging from 3E-7 to 3E-4 M is possible with the pH step titration method. In order to lower the detection limit and to improve the device-to-device reproducibility, other membrane materials may have to be investigated. Especially for selective protein detection, requiring specific receptor molecules deposited in the membrane, more hydrophilic membrane materials are probably necessary, since the hydrophobic polystyrene beads are known to adsorb easily proteins aspecifically. 

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. 

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. 

Fig. 16. Titration data for the carboxyl groups of lysozyme, at 25°C, at three ionic strengths (/). Plotted from data of Tanford and Wagner (1954).

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. 

The count of other titratable groups of lysozyme agrees with analytical data. Moreover, no variation between different preparations has been reported. 

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

Lysozyme, insulin, and catalase At high pH, irreversibly titrating Tyr s found in all three proteins Inada (1961)... 

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... 

Fullerton et al. (1986) measured H spin-lattice relaxation during dehydration of lysozyme solutions to a nearly dry state, and during rehydration of lyophilized lysozyme powder by isopiestic equilibration and, for high hydration levels, by titration with water. Breaks in the NMR response were found at 0.055, 0.22-0.27, and 1.22-1.62 h (Fig. 19 shows the two higher hydration discontinuities in slope). Estimates of the water correlation times are 10 , 2 x 10 , and 5 X 10 sec, respectively, for the three classes of water defined by the breaks. The 0.055... 

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