Measuring the p<i>K</i>/p<i>I</i> of Biomolecules Using X‑ray Photoelectron Spectroscopy

Abstract

Dissociation constants of GG–X–GG and X₅ 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, p<i>K</i> 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 p<i>K</i> values for the Asp (3.1 and 2.4), His (6.7), and Lys (11.3 and 10.6) peptides, and the p<i>I</i> 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