pK values of the ionizable groups of proteins

Abstract

The acid/base properties of proteins have been studied since 1917, when Sorensen, who first defined pH in 1909, showed that egg albumin is an ampholyte (Sorensen et al. 1917). Soon thereafter, Linderstrom-Lang recognized that the net charge on a protein would influence the ionization of individual groups, and incorporated this into the first model developed to understand the acid/base properties of a protein (Linderstrom-Lang 1924). An important contribution by Tanford and Kirkwood triggered an intense interest in the factors that determine the pK values of the ionizable groups of proteins that continues to the present day (Tanford and Kirkwood 1957; Braun-Sand and Warshel 2005). The net charge on a protein varies with pH, and is determined by the content and the pK values of the ionizable groups (Tanford 1962). Thus, the pK values of the ionizable groups are important to biochemists because they influence the structure, stability, solubility, and the many functions of proteins (Tanford 1968; Pace 1975; Fersht 1985; Matthew et al. 1985; Anderson et al. 1990; Ries-Kautt and Ducruix 1997; Shaw et al. 2001; Bartlett et al. 2002).

In early studies aimed at interpreting titration curves of proteins, Tanford (1962) introduced the term intrinsic pK (pK_int). He defined the term as the pK an ionizable group would have when the net charge on the molecule is zero. When proteins fold, the perturbations of the pKs of the ionizable groups on the surface of the protein from the pK_int values are usually small, and are determined mainly by charge–charge interactions with other ionizable groups (Laurents et al. 2003). However, if these groups are partially or fully buried in the protein interior, large positive and negative perturbations often occur, and it is important that we understand why (Schutz and Warshel 2001). Experimental studies of these perturbations have been reported by several groups (see, for example, Garcia-Moreno et al. 1997; Giletto and Pace 1999; Dwyer et al. 2000; Edgcomb and Murphy 2002; Forsyth et al. 2002; Harris and Turner 2002; Laurents et al. 2003; Horng et al. 2005; Pujato et al. 2005; Trevino et al. 2005), and theoretical studies have shown that the perturbations result primarily from the Born effect, charge–charge interactions, and hydrogen bonding (see, for example, Schutz and Warshel 2001; Fitch et al. 2002; Georgescu et al. 2002; Braun-Sand and Warshel 2005).

Studies of pK perturbations in proteins require accurate, unperturbed pK values. Historically, these pK values were determined using small molecules or peptides as models, and direct techniques such as potentiometric titration or indirect techniques such as NMR to monitor ionization. In Table ​Table11 we summarize the values commonly used in the literature. These measurements were made at various temperatures, and some were corrected for electrostatic interactions and/or ionic strength effects and some were not (see Table ​Table11 of Tanford 1962 for a description of these corrections). Most researchers today use the pK_int values from Nozaki and Tanford (N&T) (Nozaki and Tanford 1967), which are given in column 3 of Table ​Table1.1. Our goal in this paper is to provide accurate pK measurements using a better model system than any of those used previously.

Table 1.

pK values for the ionizable groups in proteins from the literature

These pK values were measured at various temperatures, as noted below. Typically, the error in the measurements is ±0.1–0.2. In some studies no salt was present, and in some the ionic strength was not given. We have not corrected any of the values given in the original articles. Usually the errors in measuring the pK values are greater than these corrections (Tanford 1962).

^{Determined using various model compounds at 25°C, as described in Table} ​Table11 in Chapter 20 of Cohn and Edsall (1943).

^{Determined using various model compounds at 25°C, as described in Table 3 of} Nozaki and Tanford (1967).

^{Determined with Gly-Gly-X-Gly-Gly pentapeptides by C NMR at 26°C, except for Tyr, which was determined at 33°C (see} Gurd et al. 1972; Keim et al. 1973). Both termini of the peptides were not blocked.

^{Determined in Gly-Gly-X-Ala tetrapeptides by C NMR at 35°C (see} Richarz and Wuthrich 1978). Both termini of the peptides were not blocked.

^{From Creighton's textbook on protein biochemistry that references some of the values from the previous columns (}Creighton 1993).

In this paper we report pK values for the ionizable groups of proteins measured by potentiometric titration using alanine based pentapeptides in 0.1 M KCl at 25°C. We believe these peptides provide the most reasonable model for determining the pK values for the ionizable groups in a protein. We compare our measurements to the N&T pK_int values given in Table ​Table1.1. These results will be useful to those interested in pK perturbations in proteins, and to those who use theory to understand the pK values of ionizable groups in proteins.

These pK values were measured at various temperatures, as noted below. Typically, the error in the measurements is ±0.1–0.2. In some studies no salt was present, and in some the ionic strength was not given. We have not corrected any of the values given in the original articles. Usually the errors in measuring the pK values are greater than these corrections (Tanford 1962).

^{Determined using various model compounds at 25°C, as described in Table} ​Table11 in Chapter 20 of Cohn and Edsall (1943).

^{Determined using various model compounds at 25°C, as described in Table 3 of} Nozaki and Tanford (1967).

^{Determined with Gly-Gly-X-Gly-Gly pentapeptides by C NMR at 26°C, except for Tyr, which was determined at 33°C (see} Gurd et al. 1972; Keim et al. 1973). Both termini of the peptides were not blocked.

^{Determined in Gly-Gly-X-Ala tetrapeptides by C NMR at 35°C (see} Richarz and Wuthrich 1978). Both termini of the peptides were not blocked.

^{From Creighton's textbook on protein biochemistry that references some of the values from the previous columns (}Creighton 1993).

^{From the model compound data in Table 3 of} Nozaki and Tanford (1967).

^{The pK values of the ionizable groups in the alanine pentapeptides used in this work measured in 0.1 M NaCl at 25°C. The errors are the standard deviations from the average of three independent experiments for the α-carboxyl, Glu, α-amino, and Tyr; and two independent experiments for Asp, His, Cys, and Lys. For all of the titrations, the error in the pK value from fitting the experimental data to Equations} 1 or 2 was <0.03.

Acknowledgments

Footnotes