Measuring the p<i>K</i>/p<i>I</i> of Biomolecules Using X‑ray
Photoelectron Spectroscopy
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Abstract
Dissociation constants of GG–X–GG
and X<sub>5</sub> 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<sup>–1</sup> 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