Proteins are abundant and in most contexts where they arepresent interfaces of different kinds are found. Interfacesinfluence protein function, and indeed many proteins areincapable of performing their proper functionwithoutthe presence of an interface. The object of thisthesis has been to study the behaviour of globular proteins atthe solid/liquid interface and the effect their presence has onthe interactions between macroscopic surfaces. This ismotivated by the many questions about the different drivingforces for adsorption which arise when the kinetics of proteinadsorption is modelled. To this end, two main techniques formeasuring the interaction between macroscopic surfaces havebeen employed: An interferometric surface force technique inwhich the substrate surfaces have been negatively chargedmuscovite mica, and a non-interferometric technique which makesuse of glass spheres, also negatively charged, as thesubstrate.
The observations can be summarized as follows. The additionof a positively charged globular protein, such as the hen eggwhite lysozyme used in this study, leads to an almost completeneutralization of the high negative surface charge of mica, atthe higher concentrations studied (>0.02 mg/ml). Theadsorption takes place with a simultaneous regulation of theadsorption of small ions to the surface. This demonstrates thatone important driving force for adsorption of globular proteinsto oppositely charged surfaces is electrostatic interactions.The measured thickness of the adsorbed protein layers providesevidence that the protein retains its structure when adsorbingsince this value is comparable to the known dimensions of theprotein in solution.
Employing two variants of bacteriophage T4 lysozyme, thewild type and one synthetic mutant, it is shown that thestructurally less stable mutant protein loses its tertiarystructure upon adsorption. The more stable wild type behavessimilarly to the structurally stable hen egg white lysozyme.The interactions between adsorbed layers, however, are rathersimilar for the two T4 lysozyme variants, despite thedifferences in their adsorbed structure. The most markeddifference is a strong attraction between a mutant layer and abare negatively charged substrate surface, indicating thestrong affinity between the surface denatured protein and thesurface. The effect of substrate on the interaction has alsobeen studied and the lower charge of a glass surface resultedin a considerably different adsorption behaviour.
If an anionic surfactant is introduced to preadsorbed layersof lysozyme on mica in protein-free solutions, the surfactantadsorbs to the protein layers resulting in an increasedinterfacial charge as evidenced by an increase in the longrange repulsion between such surfaces. At a concentration justbelow thecmcthe complex formed between the protein and thesurfactant starts to change its conformation. When the chargeof the complex becomes comparable to that of the surface,desorption takes place.
Keywords:Globular proteins, protein adsorption,desorption, elutability, structural stability, conformationchanges, surface denaturation, lysozyme, surfactant, SDS, mica,glass surface, surface forces, electrostatic forces
Institutionen för fysikalisk kemi , 1998. , 41 p.