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A new biotin derivative functionalized by an electropolymerizable pyrrole group has been synthesized. The electrooxidation of this biotin pyrrole has allowed the formation of biotinylated conducting polypyrrole films in organic electrolyte. Gravimetric measurements based on a quartz crystal microbalance, modified by the biotinylated polymer, revealed an avidin-biotin-specific binding at the interface of polymer-solution. The estimated mass increase corresponded to the anchoring of 1.5 avidin monolayers on the polypyrrole surface. In addition, the subsequent grafting of biotinylated glucose oxidase was corroborated by electrochemical permeation studies. Enzyme multilayers composed of glucose oxidase or polyphenol oxidase were elaborated on the electrode surface modified by the biotinylated polypyrrole film. The amperometric response of the resulting biosensors to glucose or catechol has been studied at +0.6 or -0.2 V vs SCE, respectively.

The catalytic activity of a glycosylphosphatidylinositol (GPI)-anchored alkaline phosphatase has been studied in Langmuir phospholipid monolayers at different surface pressures. The enzyme substrate, p-nitrophenyl phosphate, was injected into the subphase of mixed enzyme/lipid Langmuir monolayers. Its hydrolysis product was followed by monitoring the absorbance at 410 nm in situ in the monolayer subphase of the Langmuir trough. Several surface pressures, corresponding to different molecular surface densities, were attained by lateral compression of the monolayers. The morphology of the monolayers, observed by fluorescence microscopy, showed three different types of domains owing to the heterogeneous partition of the enzyme within the mixed enzyme/lipid film. The catalytic activity was modulated by the enzyme surface density, and it increased until a pressure of 18 mN/m was reached, but it decreased significantly when the equilibrium in-plane elasticity (surface compressional modulus) increased more noticeably, resulting in alterations in the interface morphology. A model for the modulation of the enzyme orientation and catalytic activity by lipid/enzyme surface morphology and enzyme surface packing at the air/liquid interface is proposed. The results might have an important impact on the comprehension of the enzymatic activity regulation of GPI-anchored proteins in biomembranes.

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