Potentiometric titration and enthalpy evaluation of horseradish peroxidase in the presence of n-dodecyl trimethylammonium bromide

Abstract

The reversible proton dissociation equilibria of horseradish peroxidase (HRP) is investigated in 100 mM NaCl solution at 25°C and over a pH range of 3.0–11.0 in the absence and presence of n-dodecyl trimethylammonium bromide (DTAB). Intrinsic dissociation constants for the carboxyl groups of acidic residues and also for the imidazole side chains of histidine residues in HRP are estimated using the Tanford expression. From the obtained titration curve the intrinsic pK a (pK a,int) of titratable groups, the electrostatic free energy ( W el), the enthalpy of unfolding (ΔH u) and especially the enthalpy of ionisation (ΔH ion) for the enzyme molecule are estimated. Also the state of ionisation is discussed in terms of ionisation enthalpy changes. Results indicate that 79% of the total carboxyl groups are present in the form of buried or masked groups, which are not exposed to titration. While in the presence of DTAB, 65% of these groups remain buried and the isoelectric pH varies from 8.8 (in the absence of DTAB) to 5.5 (in the presence of DTAB). In the absence of surfactant no conformational changes are observed in the whole pH region of the titration experiment.