RESUMO
A new coupling strategy for biomacromolecules with (3-mercaptopropyl)trimethoxysilane (3MPTMS) and 11-(triethoxysilyl)undecanal (TESU) on gold surfaces is. This immobilization protocol was utilized for the enzyme horseradish peroxidase (HRP). To study the reactions and resulting structures, PM-IRRAS measurements were performed. PM-IRRAS shows there is structure preservation of the HRP when the new coupling strategy is used in contrast to non-specific adsorption on gold. The biological activity of adsorbed and immobilized HRP was measured by the enzyme catalyzed oxidation of 3,5,3',5'-tetramethylbenzidine. Covalent immobilization of HRP on TESU film compared to physisorption of HRP shows higher enzyme activity on gold surfaces, confirming the structural preservation detected by PM-IRRAS.
Assuntos
Enzimas Imobilizadas/metabolismo , Peroxidase do Rábano Silvestre/metabolismo , Adsorção , Ativação Enzimática , Enzimas Imobilizadas/química , Ouro/química , Ouro/metabolismo , Peroxidase do Rábano Silvestre/química , Modelos Moleculares , Estrutura Molecular , Compostos de Organossilício , Silanos/química , Silanos/metabolismo , Espectrofotometria Infravermelho , Propriedades de SuperfícieRESUMO
Antifreeze proteins (AFPs) are of great importance for applications in cryomedicine or the food industry. They are frequently used to lower the freezing point by preventing the growth of larger ice crystals; thus, it is paramount to determine their thermal hysteretic characteristics. However, the experimental analysis of the thermal hysteresis-an effect that is characteristic for AFPs-remains a challenging process. An easy-to-use test method for measuring the thermal hysteresis of AFPs was developed and tested with the type III AFPs. Traditional methods that have been used until now have their disadvantages and limitations. The new measurement method described in this paper allows detection of the complete cooling, freezing, heating, and melting process in a single measurement. This makes it possible to directly determine the thermal hysteresis as a functional effect of the antifreeze proteins. Measurements of the thermal hysteresis were performed by applying ultrasound to initiate the crystallization process of the antifreeze protein solution. This ultrasound technique also allows a crystallization process to be performed at defined temperature. The demonstrated results were highly reproducible and could be clearly read off the measurement curves. As a future perspective, this enables the design of automatic test devices that can be also miniaturized.
