| Abstrakt: |
Horseradish peroxidase (HRP) has been used as a model enzyme in this study of its physical adsorption and residual enzyme activity on radio frequency glow discharge (RFGD)-treated polymers. The specific enzymatic activity of HRP adsorbed on different surfaces was assumed to be an indication of the extent of its conformational alterations on the surfaces. The surfaces studied were poly (ethylene terephthalate) (PET), polytetrafluoroethylene (PTFE), and tetrachloroethylene and tetrafluoroethylene glow discharge-treated PET, abbreviated as TCE/PET and TFE/PET. All surfaces were characterized by electron spectroscopy for chemical analysis (ESCA) and liquid contact angles in air. HRP adsorbs more strongly onto the two discharge-treated surfaces than onto the untreated polymers, as evidenced by the lower amount of HRP eluted by sodium dodecyl sulfate (SDS) from the treated polymers. For example, seventy percent of the HRP adsorbed on TCE/PET or TFE/PET remains on the surface after overnight elution with a 1% solution of SDS. In contrast, untreated PET and PTFE each retains only c. 20% of the absorbed enzyme. The enzymatic activity of HRP adsorbed on the different surfaces was studied using hydrogen peroxide (H2O2) as the substrate. HRP adsorbed on the higher energy surfaces, PET and TCE/PET, retains significantly more activity than the HRP adsorbed on the lower energy surfaces, PTFE and TFE/PET which appear to destroy rapidly almost all of the activity of HRP after it adsorbs. HRP adsorbed on TCE/PET is relatively more stable over time than HRP adsorbed on PET or free HRP in solution. (For example, only 45% of the specific enzymatic activity of HRP adsorbed on TCE/PET was lost after 3 h of storage in phosphate buffer at 37°C, while 70% of that adsorbed on PET was lost.) In summary, when HRP is adsorbed on TCE/PET, it is very tightly bound, and yet it maintains a significant fraction of its initial specific activity and also retains this activity for 3 h in phosphate buffer at 37°C. Thus, tenacious physical adsorption of proteins such as enzymes on TCE glow discharge-treated surfaces may have potential as a new method of immobilization of such molecules, for uses in biosensors, diagnostics, bioseparations, cell culture and bioreactors. |
