title: The determination of the pKa of histidine residues in proteins by Raman difference spectroscopy.
abstract: Sensitive Raman difference spectroscopy was used to monitor the protonation and deprotonation of histidine residues in apo-transferrin. We have shown previously that the behavior of small molecules and/or small molecular groups bound to proteins or other large macromolecules can be studied by Raman difference spectroscopy (Yue, K.T. et al. (1989) J. Raman Spectrosc. 20, 541-545). Using this method, we have measured the Raman difference spectra of human transferrin at different pH values with respect to pH 8.9, titrating its various histidine residues. About 12 +/- 2 of the 19 residues were titrated. The pH difference spectrum of transferrin obtained is very similar to that of histidine in solution, but with clear differences in the 1200-1400 cm-1 region. A titration curve with pKa of 6.08 +/- 0.01 fit the data of histidine in solution and a value of 6.56 +/- 0.02 was found for the average value of the 12 histidine residues inside transferrin. The technique has enough sensitivity at present to monitor a single histidine residue in a 130 kDa molecule and to determine the titration curve of one residue in a 40 kDa protein.
authors.affiliation: Physics Department, Emory University, Atlanta, GA 30322.
authors.forename: K T
authors.initials: KT
authors.lastname: Yue
authors.forename: M
authors.initials: M
authors.lastname: Lee
authors.forename: J
authors.initials: J
authors.lastname: Zheng
authors.forename: R
authors.initials: R
authors.lastname: Callender
mesh_terms: D001059:Apoproteins / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D013059:Spectrum Analysis, Raman; D014168:Transferrin / Q000737:chemistry*
chemical_list: D001059:Apoproteins; D014168:Transferrin; C028284:apotransferrin; D006639:Histidine