Imidazole group protein, titration

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... 

This same explanation clearly cannot be invoked to account for the observed pK values of imidazole groups, since these pK s differ from expectation in the wrong direction. This result should perhaps not be taken too seriously since the intrinsic pK s of protein imidazole groups are relatively imprecise. Moreover, there is some question about the expected pK of an imidazole group (Table I, h). On the other hand, the volume changes which accompany dissociation of imidazole groups, to be discussed in Section VIII, are also anomalous, and the over-all conclusion must be that some effect of protein structure on the titration of imidazole groups has so far been overlooked in theoretical treatments of the problem. 

It should be noted that four of the anomalous imidazole groups are the four groups by which the heme iron atoms are attached to the protein. These cannot be titrated as long as the hemes remain attached. The other groups must be simply buried in the interior. Such groups occur in myoglobin (see below), as well as in hemoglobin. 

Understanding of the metal-protein interaction between zinc and insulin has recently been enhanced by studies of titration curves. Crystalline insulin as normally prepared contains a small amount of zinc, and moreover is insoluble between pH 4 and 7, where pH drifts are also observed. Fred-ericq (1954) titrated two fractions of insulin which differed in their solubility at pH 8, and reported that two imidazole groups which dissociate near pH 7 in the soluble form are not evident in the insoluble fraction. 

In view of the anomalous behavior of these amino acid residues, the question arises whether these titrations are reversible. Figure 8 shows that reversibility could not be demonstrated on back titration from pH 11.5 with acid. The lower solid curve represents the forward titration, the upper one is the back titration, whereas the dashed curves are those of the second forward and back titration. It is clear that there is an increase in the number of titratable groups on back titration and the apparent pK of the imidazole residues has now been shifted to a lower value—e.g., to pH 6.9. Thus the irreversibility of these titrations can be taken as a reflection that an irreversible configurational change occurs on exposure of the protein to alkaline pH. 

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