Calorimetric determination of the enthalpy of ionization of oxidized and reduced horse heart cytochrome c

Abstract

SynopsisThe heats of ionization of protons, AH,, of oxidized and reduced horse heart cytochrome c in 0.15M KC1 a t 20°C were determined using a titration calorimeter which simultaneously afforded the potentiometric titration curve. Reproducibility of the thermal titrations is within 2%, and evaluation of the heats observed after the heat loss corrections is estimated to be within 5%. A single titration of oxidized cytochrome c from pH 11 to 3 is in excellent agreement with the thermal titration of this protein obtained with flow calorimetry. The thermal titration, however, is not reversible, due in part to the loss of titratable group(s) in this pH region and to the heat contribution of the acid and alkaline conformational changes which occur. Although of lesser magnitude, the reduced form also indicates similar thermal transitions. These differences are due solely to conformational contributions to the thermal process, since the potentiometric curves are reversible. The nature of the irreversibility for oxidized cytochrome c appears to involve the loss of a group with pK' 8.9 and the shift of two groups from pK' 5.6 to 4.8. Thermal difference curves for this process indicate that heats of -7.8 and -24.1 kcal/mol are liberated which are centered at pH 9.3 and 3.9, respectively.

Are the electrospray mass spectra of proteins related to their aqueous solution chemistry?

“…Note that the pK in t is lower than that reported by Nozaki and Tanford [31], and lower than that observed for cytochrome c as described in the previous section. This difference, however, is not significant, The intrinsic pKas reported by Marini and Martin [39] were 3.8 for glutamic acid and 3.0 for aspartic acid, in a model of the titration of cytochrome c in which the electrostatic repulsion factor w (eq 2) was determined to be zero. Figure 3 illustrates the comparison of the distribution of multiply charged ions in the electrospray spectra of myoglobin, and the distribution of preformed protein ions in the bulk solution.…”