ABSTRACT
This report demonstrates the feasibility of radiation grafting for the preparation of polymer layers functionalised with short peptide ligands which promote cell adhesion. Thermoresponsive poly [tri(ethylene glycol) monoethyl ether methacrylate] (PTEGMA) layers were synthesised on a polypropylene substrate by post-irradiation grafting. A cell adhesion moiety, the CF-IKVAVK peptide modified with a methacrylamide function and a fluorescent label were introduced to the surface during the polymerisation process. The amount of CF-IKVAVK was easily controlled by changing its concentration in the reaction mixture. The changes in the surface composition, morphology, philicity and thickness at each step of the polypropylene functionalisation confirmed that the surface modification procedures were successful. The increase in environmental temperature above the cloud point temperature of PTEGMA caused a decrease in surface philicity. The obtained PTEGMA and PTEGMA-peptide surfaces above TCP were tested as scaffolds for fibroblast sheet culture and temperature induced detachment.
Subject(s)
Peptides/chemistry , Polyethylene Glycols/chemistry , Radiation , Temperature , Amino Acid Sequence , Chromatography, High Pressure Liquid , Fibroblasts/cytology , Humans , Methacrylates/chemical synthesis , Methacrylates/chemistry , Microscopy, Atomic Force , Peptides/chemical synthesis , Polyethylene Glycols/chemical synthesis , Polymethacrylic Acids/chemical synthesis , Polymethacrylic Acids/chemistry , Skin/cytology , Solid-Phase Synthesis Techniques , Spectrometry, Mass, Electrospray Ionization , Spectrometry, Mass, Secondary Ion , Spectroscopy, Fourier Transform Infrared , Surface PropertiesABSTRACT
A spectrophotometric assay for glucosidase I using the synthetic trisaccharide alpha-D-Glc 1-->2 alpha-D-Glc 1-->3 alpha-D-Glc-O(CH2)8COOCH3 is reported. The terminal glucose is released from the substrate by the enzyme and quantitated using glucose oxidase, peroxidase, and o-dianisidine. The trisaccharide is specific for glucosidase I and provides all the necessary structural features for correct interaction in the enzyme active site. The utility of the assay for monitoring enzyme activity during isolation and for use in kinetic and inhibition studies (i.e., with 1-deoxynorjirimycin) is demonstrated.
