ABSTRACT
The conformation of adsorbed proteins has a significant influence on the bioactivity of the proteins or subsequent processes like biofouling, implant rejection, and others. The design of protein-contacting materials requires a detailed knowledge of protein conformation in the adsorbed state as a function of the physical and chemical properties of the sorbent surface. In addition to the protein biological function, it has been shown that conformational changes are a crucial factor in describing the adsorption process. Nowadays, there are many experimental methods available to directly access information on the conformation of adsorbed proteins. Among them, one of the most prominent techniques is optical spectroscopy. In this chapter we review the principles of optical spectroscopy as they apply to a population of protein molecules adsorbed at an interface. We start by reviewing the methods for enhancing the signal from the interfacial molecules and follow with a short description of three spectroscopic techniques most often used in protein adsorption studies: fluorescence, infrared absorbance, and circular dichroism spectroscopy.*
A. Interaction of Protein Molecules with Light The interaction between light and molecules represents one of the fundamental problems in quantum optics [4]. It is impossible to visualize a single light quantum, a photon, propagating at the speed of light through space because of the inherent uncertainty about the photon's position. Nevertheless, one can imagine a stream of photons moving through space: a light beam. The electromagnetic field associated
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with each proton will induce a time-dependent charge redistribution of a finite dimension in its surrounding. If the space through which the stream of photons moves is occupied by a protein molecule, the entities interacting with the photons' electromagnetic field may be a covalent bond between two atoms, a molecular orbital, some delocalized states, or some vibrational or rotational modes involving a part or whole protein molecule. The complexity of these interactions is simplified by modeling the molecule as a collection of dipoles which interact with the propagating electromagnetic field [5]. It will take the electromagnetic field traveling at the speed of light a mere 3 X 10-18 s to zip through a protein of average size. And yet the effect of the traveling electromagnetic field on the surrounding dipoles in the molecule produces experimentally measurable quantities: changes in transmitted light intensity, polarization, and rotation and differences of energy and angle of the scattered light. Measuring and interpreting these changes falls in the realm of optical spectroscopy.
