Tertiary structure changes in albumin upon surface adsorption observed via fourier transform infrared spectroscopy.

A nondestructive Fourier transform infrared (FTIR) spectroscopy assay, amenable to exploring a wide range of proteins and polymers, is used to measure changes in the tertiary structure of bovine serum albumin (BSA) adsorbed to three surfaces: gold, polystyrene (PS), and poly(D,L-lactic acid) (PDLLA). Tertiary structural analysis is important because typical secondary structural analysis (FTIR and CD) is not always sensitive enough to distinguish between the sometimes subtle protein structural changes caused by adsorption. The polymers are spin-coated onto a gold surface, exposed to protein, and then immersed in a deuterated buffer solution to probe the protein's tertiary structure before the sample is removed from its aqueous environment. Infrared band intensities, related to the exchange of amide hydrogen for deuterium (HDX), as a function of the immersion time in deuterated buffer, are used to determine the extent of amide solvent exposure. Analysis of the results in terms of a single exponential decay shows that enough amides undergo a measurable amount of exchange in 60 min to quantify relative changes in BSA solvent exposure on different surfaces. In addition, substantial fractions undergo HDX at a rate too fast or too slow to be followed with our experimental protocol. The proportions of these quickly and slowly exchanging amide groups also provide information about relative changes in the BSA structure on different surfaces. Adsorption was found to increase the extent of HDX over that observed for BSA in solution, consistent with surface-induced unfolding and a loss of tertiary structure. Changes in HDX were found to be more sensitive to which surface was absorbing the protein than the typical FTIR secondary structural analysis obtained from fitting the amide I band. HDX was greatest for BSA adsorbed to the surface of PDLLA and least in the case of BSA adsorbed to gold, which indicates the greatest and least degree of unfolding, respectively.