Methoden zur Immobilisierung von Proteinen auf Polyurethan- und Goldoberflächen und ihr Einfluss auf Konformation und Aktivität der Proteine

Abstract

In recent years anti-freeze proteins became the focus of interest for materials science due to their ice-crystall-growth inhibiting properties, recrystallisation properties and ice-crystall structuring properties. The transfer of these properties to surfaces by means of a molecular biomimetic approach is the challenge as well as motivation of this work. Here, the molecular bionic approach is based on chemical immobilization methods of proteins to solid surfaces. Thus, the first question of this work can be stated; upon the understanding interrelationships of the activity of immobilized and dissolved antifreeze protein type III (AFP type III). To obtain an answer to this question, the activity of AFP type III in case of its dissolved state was determined by sonocrystallisation, a newly developed measuring device, and differential scanning calorimetry. The activity of anti-freeze proteins on the surface was analyzed by con-ducting a rime test and a frost-adhesion test in the rime chamber. As solid surfaces acted in the present work a high-energy gold surface on the one hand and a low-energy polyurethane surface on the other hand. Due to the chemical inertness of the polyurethane surface com-pressed-air plasma activation was performed. The purpose of the atmospheric pressure plasma activation was to introduce the functional groups necessary for immobilization. Generally, it is known from the literature that proteins in face boundaries, such as protein-water interface [1], [2], [3] and protein-solid interface [4], [5], [6], are subject to deconformation, also known as denaturation, due to intermolecular interactions. For this rea-son, the effect of the protein-solid interface on both, the conformation as well as the bioactivi-ty, represents the most important aspect of this work. In this context, the second question of this study refers to understanding the relationships between the conformational change and the activity of proteins after immobilization. The effects of immobilization on the confor-mation have been successfully studied by vibrational spectroscopy using horseradish peroxi-dase and anti-freeze protein type III. To verify a possible correlation between surface-induced conformational change and activity of horseradish peroxidase, activity tests were performed on horseradish peroxidase-solid interphase using the colorless chromogen 3,3',5,5'-tetramethylbenzidine.