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Measuring Hydrogen Deuterium Exchange in Protein Monolayers



Jack R. Smith, Marcus T. Cicerone, Curtis Meuse


A non-destructive infrared (IR) spectroscopy assay to measure hydrogen-deuterium exchange (HDX) in surface-adsorbed protein monolayers has been developed. The procedure can be used to measure HDX for different proteins on a variety of materials applied to gold-coated substrates. Adsorbed protein HDX is quantified by exposing the protein to a 50 % deuterated (i.e., 50 - 50 % D2O-H2O) 0.01 M NaPO4 buffer solution and then measuring the ratio (f) of the intensities of the amide II (N-H in plane bend) and amide I (amide C=O stretch) bands in the IR reflection spectrum.If collected as a function of time, f can follow the kinetics of the exposure of protein amides to solvent. This measure of amide exposure can then be related to subsequent measurements of biological response for applications in fields such as medical implants, biosensors and drug delivery. HDX kinetics have been obtained for Bovine Serum Albumin (BSA) in solution and adsorbed to gold surfaces. Adsorption to gold was found to increase the rate of change of f over that observed for BSA in solution, consistent with an increase in the exposure of albumin amide groups upon adsorption. We also show that BSA adsorbs to the surface of gold in multilayers and that the increase in amide exposure is present only in the first adsorbed monolayer.
Analytical Chemistry


hydrogen deuterium exchange, infrared spectroscopy, protein adsorption


Smith, J. , Cicerone, M. and Meuse, C. (2008), Measuring Hydrogen Deuterium Exchange in Protein Monolayers, Analytical Chemistry (Accessed April 17, 2024)
Created October 16, 2008