The adsorption of trypsin on hydrophilic and hydrophobic surfaces. In situ structural characterization of the enzyme in the adsorbed state

The adsorption of trypsin onto polystyrene and silica surfaces was investigated by reflectometry, spectroscopic methods, and atomic force microscopy (AFM). The affinity of trypsin for the hydrophobic polystyrene surface was higher than that for the hydrophilic silica surface, but steady-state adsorbed amounts were about the same at both surfaces. The conformational characteristics of trypsin immobilized on silica and polystyrene nanospheres were analyzed in situ by circular dichroism and fluorescence spectroscopy. Upon adsorption the trypsin molecules underwent structural changes at the secondary and tertiary level, although the nature of the structural alterations was different for silica and polystyrene surfaces. AFM imaging of trypsin adsorbed on silica showed clustering of enzyme molecules. Rinsing the silica surface resulted in 20% desorption of the originally adsorbed enzyme molecules. Adsorption of trypsin on the surface of polystyrene was almost irreversible with respect to dilution. After adsorption on silica the enzymatic activity of trypsin was 10 times lower, and adsorbed on polystyrene the activity was completely suppressed. The trypsin molecules that were desorbed from the sorbent surfaces by dilution with buffer regained full enzymatic activity

Sweet brushes and dirty proteins

We studied the protein repellency of dextran brushes. Dextran was grafted to a polystyrene surface in a broad range of grafting densities using polystyrene - dextran block copolymers and the Langmuir - Blodgett deposition technique. Ellipsometry measurements confirmed a successful transfer of the dextran brush from the air - water interface to the polystyrene surface. Water contact-angle measurements validated the presence of the dextran layer at the surface. At grafting densities = 0.20 nm(-2), a homogeneous dextran brush is observed. Adsorption studies of BSA and trypsin, using optical reflectometry, showed that adsorbed amounts at the heterogeneous coating (<0.20 nm(-2)) is only slightly lower, if at all, than at the bare polystyrene surface. Beyond 0.20 nm(-2), a drastic decrease in adsorbed amount was observed, due to excluded volume interactions between the protein and the homogeneous dextran brush. Almost complete protein repellency could be reached at high grafting densities. Comparison with adsorption studies of PEO brushes indicated that dextran brushes do not outperform PEO brushes in suppressing protein adsorption

Vergleichende Bewertung vorhandener Schnelltestverfahren in der Wasseranalytik

TIB Hannover: RN 8422(1989,41) / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekSIGLEDEGerman