Ionization behavior of proteins. II. Chymotrypsinogen and its group‐modified derivatives: Study of their ionization characteristics by potentiometric and calorimetric titration

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Abstract
Chymotrypsinogen, nitrated chymotrypsinogen (two of the four tyrosyls nitrated), acetylated chymotrypsinogen (all amino groups blocked), and nitrated‐acetylated chymotrypsinogen were titrated as f(pH) in an isoperibolic calorimeter at 20°C. After appropriate correction and reduction of both the potentiometric and thermal titration data, the parameters N (ionizable groups per group‐set), pK′, and ΔHi (heat of ionization) were evaluated using the iterative curve‐fitting algorithm of the MLAB computer program. The pK′ parameters so obtained for the two normally ionizing tyrosyl groups in chymotrypsinogen and the two nitrated tyrosyl groups in the nitrated proteins essentially agreed with the results of spectral titration. Excellent fits to all data could be obtained using evaluated parameter sets of N and pK′ for the potentiometric titration data (groups vs pH plots) and N, pK′, and ΔHi sets for the calorimetric data (total heat vs pH plots). The invocation of electrostatic interaction effects was not required to explain the data satisfactorily, despite the differences in charge number and type among the four proteins. Rather, the data can be represented by series expressions of the mass‐action law. Using all information, viz., the consequences of functional group modification, the downscale shift in tyrosyl group pK's on nitration, and the numerical values of the evaluated N, pK′, and ΔHi parameter sets for all proteins, the chemical identity of the various classes of group sets can be assigned with reasonable assurance.