Incorporation of alkaline phosphatase into layer-by-layer polyelectrolyte films on the surface of affi-gel heparin beads: physicochemical characterization and evaluation of the enzyme stability.
The preparation of functionalized beads in the micrometer size range that can be used to probe the action of immobilized biomolecules on cell cultures during controlled periods of time is of fundamental importance in cell biology. However, the preparation and characterization of such particles is tedious because of their fast sedimentation. It is hence difficult to prepare such beads in a reproducible manner. This highlights the need to prepare an important batch of functionnalized particles and to store them under conditions where the loss of biological activity is minimized. The aim of this paper was to immobilize alkaline phosphatase (AP) as a model enzyme on the surface of Affi-gel heparin beads functionnalized by means of a layer-by-layer (LBL) film made of poly-l-glutamic (PGA) acid and poly-l-lysine (PLL). The enzyme has been adsorbed either on the top of the LBL film or embedded under five polyelectrolyte layers. When embedded, the enzyme was not released in buffer and retained more than 30% of its initial activity after 3 months of storage at 4 degrees C. However, when the enzyme was adsorbed on top of the LBL film, about 80% of the adsorbed enzyme was released in the buffer after a few days of storage. Longer storage did not lead to any further desorption and the remaining enzyme displayed the same evolution of its activity with time as the embedded enzyme. The time evolution of the enzyme activity on the beads is compared with that in solution alone and in the presence of PGA and PLL separately.
Derbal
Lylia
L
Institut Charles Sadron, Centre National de la Recherche Scientifique, Unité Propre 22, 6 rue Boussingault, 67083 Strasbourg Cedex, France.
Lesot
Hervé
H
Voegel
Jean Claude
JC
Ball
Vincent
V
D000469:Alkaline Phosphatase / Q000737:chemistry; D015374:Biosensing Techniques / Q000379:methods*; D003432:Cross-Linking Reagents; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry*; D006493:Heparin; D008863:Microspheres; D011099:Polyglutamic Acid; D011107:Polylysine; D011108:Polymers / Q000737:chemistry
D003432:Cross-Linking Reagents; D004800:Enzymes, Immobilized; D011108:Polymers; D011107:Polylysine; D011099:Polyglutamic Acid; D006493:Heparin; D000469:Alkaline Phosphatase