4.
A linearization method based on modified Gran functions, and a general nonlinear regression program were used to study potentiometric
titration curves of denatured ovalbumin and lysozyme in 6 mol L
–1 guanidine hydrochloride medium with the aim of determining the ionizable species. With both numerical techniques it was possible
to determine the sum of the carboxylic groups, the imidazol, the α-amine, and the sum of ɛ-amine, phenolic and sulfhydryl
groups, if the protein is completely denatured, and assumes a randomly coiled conformation. A total of 87.8 ± 2.5 and 20.7
± 0.6 groups per mol were determined in the ovalbumin and lysozyme, respectively. These values are very close to the 88 and
21 groups expected by aminoacid composition of both proteins, indicating that all ionizable groups were exposed to the solvent.
For ovalbumin the distribution of groups was very similar to that expected by the aminoacid composition, but for lysozyme
some anomalies were observed, suggesting the existence of interactions between ionizable groups, altering the dissociation
constants.
Received: 9 December 1996 / Revised: 27 February 1997 / Accepted: 4 March 1997
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