The molar absorption coefficient, E, of a protein is usually based on concentrations measured by dry weight, nitrogen, or amino acid analysis. The studies reported here suggest that the Edelhoch method is the best method for measuring E for a protein. (This method is described by Gill and von Hippel [1989, Anal Biochem 182:319-3261 and is based on data from Edelhoch [ , Biochemistry 6:1948.) The absorbance of a protein at 280 nm depends on the content of Trp, Tyr, and cystine (disulfide bonds). The average E values for these chromophores in a sample of 18 well-characterized proteins have been estimated, and the E values in water, propanol, These ~(280) values are quite reliable for proteins containing Trp residues, and less reliable for proteins that do not. However, the Edelhoch method is convenient and accurate, and the best approach is to measure rather than predict E .
We tabulated 541 measured pK values reported in the literature for the Asp, Glu, His, Cys, Tyr, and Lys side chains, and the C and N termini of 78 folded proteins. The majority of these values are for the Asp, Glu, and His side chains. The average pK values are Asp 3.5 6 1.2 (139); Glu 4.2 6 0.9 (153); His 6.6 6 1.0 (131); Cys 6.8 6 2.7 (25); Tyr 10.3 6 1.2 (20); Lys 10.5 6 1.1 (35); Cterminus 3.3 6 0.8 (22) and N-terminus 7.7 6 0.5 (16). We compare these results with the measured pK values of these groups in alanine pentapeptides, and comment on our overall findings.
We have used potentiometric titrations to measure the pK values of the ionizable groups of proteins in alanine pentapeptides with appropriately blocked termini. These pentapeptides provide an improved model for the pK values of the ionizable groups in proteins. Our pK values determined in 0.1 M KCl at 25°C are: 3.67 6 0.03 (a-carboxyl), 3.67 6 0.04 (Asp), 4.25 6 0.05 (Glu), 6.54 6 0.04 (His), 8.00 6 0.03 (a-amino), 8.55 6 0.03 (Cys), 9.84 6 0.11 (Tyr), and 10.40 6 0.08 (Lys). The pK values of some groups differ from the Nozaki and Tanford (N&T) pK values often used in the literature: Asp (3.67 this work vs. 4.0 N&T); His (6.54 this work vs. 6.3 N&T); a-amino (8.00 this work vs. 7.5 N&T); Cys (8.55 this work vs. 9.5 N&T); and Tyr (9.84 this work vs. 9.6 N&T). Our pK values will be useful to those who study pK perturbations in folded and unfolded proteins, and to those who use theory to gain a better understanding of the factors that determine the pK values of the ionizable groups of proteins.Keywords: pK values; protein ionizable groups; pH titration; peptide model compounds 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 Pac...
The structure, stability, solubility, and function of proteins depend on their net charge and on the ionization state of the individual residues. Consequently, biochemists are interested in the pK values of the ionizable groups in proteins and how these pK values depend on their environment. We review what has been learned about pK values of ionizable groups in proteins from experimental studies and discuss the important contributions they make to protein stability and solubility.
The net charge and isoelectric pH (pI) of a protein depend on the content of ionizable groups and their pK values. Ribonuclease Sa (RNase Sa) is an acidic protein with a pI ס 3.5 that contains no Lys residues. By replacing Asp and Glu residues on the surface of RNase Sa with Lys residues, we have created a 3K variant (D1K, D17K, E41K) with a pI ס 6.4 and a 5K variant (3K + D25K, E74K) with a pI ס 10.2. We show that pI values estimated using pK values based on model compound data can be in error by >1 pH unit, and suggest how the estimation can be improved. For RNase Sa and the 3K and 5K variants, the solubility, activity, and stability have been measured as a function of pH. We find that the pH of minimum solubility varies with the pI of the protein, but that the pH of maximum activity and the pH of maximum stability do not.
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