title: Hyaluronidase behavior at the air/liquid and air/lipid interfaces and improved enzymatic activity by its immobilization in a biomembrane model.
abstract: Bovine testicular hyaluronidase (BT-HAase), a tetrameric enzyme responsible for randomly hyaluronic acid catalytic hydrolysis, was successfully immobilized on Langmuir-Blodgett films prepared with the sodium salt of dihexadecylphosphoric acid, (DHP-Zn(II)) ending with dipalmitoylphosphatidylcholine, DPPC. Data of protein adsorption at the air-liquid interface by means of pendant drop shape analysis and interaction of the protein with Langmuir monolayers of DPPC, using a Langmuir trough, have provided information about the conditions to be used in the protein immobilization. The dynamic surface pressure curves obtained from pendant drop experiments for the enzyme in buffer solutions indicate that, within the range of concentration investigated in this study, the enzyme exhibits the largest induction time at 5 μg L(-1) attributed to diffusion processes. Nevertheless, it seems that, at this concentration, the most probable conformation should be the one which occupies the smallest area at π→0. The surface pressure (π) area curves obtained for BT-HAase and mixed DPPC-BT-HAase monolayers reveal the presence of the enzyme at the air-lipid interface up to 45 mN m(-1). Tests of enzymatic activity, using hyaluronic acid, HA, as the substrate, showed an increase of activity compared to the homogeneous medium. A simplified model of protein insertion into the lipid matrix is used to explain the obtained results.
authors.affiliation: Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil.
authors.forename: Douglas Santos
authors.initials: DS
authors.lastname: Monteiro
authors.forename: Thatyane Morimoto
authors.initials: TM
authors.lastname: Nobre
authors.forename: Maria Elisabete Darbello
authors.initials: ME
authors.lastname: Zaniquelli
mesh_terms: D015060:1,2-Dipalmitoylphosphatidylcholine / Q000737:chemistry*; D000327:Adsorption; D000388:Air; D000818:Animals; D002417:Cattle; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism; D006820:Hyaluronic Acid / Q000737:chemistry / Q000378:metabolism; D006821:Hyaluronoglucosaminidase / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D013050:Spectrometry, Fluorescence
chemical_list: D004800:Enzymes, Immobilized; D015060:1,2-Dipalmitoylphosphatidylcholine; D006820:Hyaluronic Acid; D006821:Hyaluronoglucosaminidase