Measuring the pK/pl of Biomolecules Using X-ray Photoelectron Spectroscopy

摘要

Dissociation constants of GG-X-GG and X_5 peptides (X = G, D, H, or K), and bovine albumin (BSA) and fibronectin (FN) were measured by X-ray photoelectron spectroscopy (XPS) in ultrahigh vacuum at room temperature. The biomolecules were deposited on Au substrates by drying 2.0 μL drops of 1.0 μg μL~(-1) stock solutions in 100 mM sodium phosphate buffers (pH 1-12) at room temperature. Because of the ～+1.3 eV shift in binding energy (BE) of protonated amines, pK values of basic amino acids were calculated by plotting the fraction of protonated amines as a function of solution pH. Similarly, the BE of carboxyl groups shifted ～-1.3 eV upon deprotonation. While C 1s spectra were convoluted by the multiple chemical states of carbon present in the samples, the ratio of the C 1s components centered at BE = 289.0 ± 0.4 and BE = 287.9 ± 0.3 proved to reliably assess deprotonation of carboxyl groups. The pK values for the Asp (3.1 and 2.4), His (6.7), and Lys (11.3 and 10.6) peptides, and the pI of BSA (4.8) and FN (5.7), were consistent with published values; thus, these methods could potentially be used to determine the dissociation constants of surface-bound biomolecules.