Hydrogen-ion titrations of amino acids and proteins in solutions containing concentrated electrolyte
- Technische Universitaet Muenchen (Germany)
- Lawrence Berkeley Lab., CA (United States)
This report describes a first attempt to quantify the net charge as a function of solution pH for lysozyme and {alpha}-chymotrypsin at 0.1 M, 1.0 M and 3.0 M ionic strength, (IS). The calculations are based on the residue (titratable group) pK{sub a}`s in the amino-acid sequence of the protein. To determine these pK{sub a}`s, a simple theory was used which assumes that the pK{sub a}`s are independent from each other in the protein and are equal to their pK{sub a} values in free amino-acid solution (Independent-Site Theory, IST). Residue pK{sub a}`s were obtained from amino-acid hydrogen-ion titrations at three different KCl concentrations corresponding to 0.1M, 1.0M and 3.0M ionic strength. After construction of a suitable apparatus, the experimental procedure and data reduction were computerized to perform a large number of titrations. Most measured pK{sub a}`s showed high reproducibility (the difference of pK{sub a} values observed between two experiments was less than 0.05). For IS = 0.1M, observed pK{sub a}`s agreed with literature values to within a few hundredths of a pH unit. Furthermore, the ionic-strength dependence of the pK{sub a}`s followed the trends reported in the literature, viz. pK{sub a} values decrease with increasing ionic strength until they reach a minimum at about IS = 0.5M. At still higher IS, pK{sub a}`s increase as the ionic strength rises to 3M. The known pK{sub a}`s of all titratable groups in a protein were used with the IST to give a first approximation of how the protein net charge varies with pH at high ionic strength. A comparison of the titration curves based on the IST with experimental lysozyme and {alpha}-chymotrypsin titration data indicates acceptable agreement at IS = 0.1M. However, comparison of measured and calculated titration curves at IS = 1M and IS = 3M indicates only quantitative agreement.
- Research Organization:
- Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
- Sponsoring Organization:
- USDOE, Washington, DC (United States)
- DOE Contract Number:
- AC03-76SF00098
- OSTI ID:
- 10124599
- Report Number(s):
- LBL-36511; ON: DE95008356; TRN: 95:002677
- Resource Relation:
- Other Information: PBD: Dec 1994
- Country of Publication:
- United States
- Language:
- English
Similar Records
Characterization and determination of titratable groups of proteins by linearization of titration curves. II. Application to lysozyme
Unusual chemical properties of N-terminal histidine residues of glucagon and vasoactive intestinal peptide