title: Savinase proteolysis of insulin Langmuir monolayers studied by surface pressure and surface potential measurements accompanied by atomic force microscopy (AFM) imaging.
abstract: The mechanism of the enzymatic action of Savinase on an insulin substrate organized in a monolayer at the air-water interface was studied. We followed two steps experimental approach classical surface pressure and surface potential measurements in combination with atomic force microscopy imaging. Utilizing the barostat surface balance, the hydrolysis kinetic was followed by measuring simultaneously the decrease in the surface area and the change of the surface potential versus time. The decrease in the surface area is a result of the random scission of the peptide bonds of polypeptide chain, progressively appearance of amino acid residues, and their solubilization in the aqueous subphase. The interpretation of the surface potential data was based on the contribution of the dipole moments of the intact and broken peptide groups which remain at the interface during the proteolysis. An appropriate kinetic model for the Savinase action was applied, and the global kinetic constant was obtained. The application of the AFM revealed the state of the insulin monolayers before and after the Savinase action. The comparison of the topography of the films and the roughness analysis showed that insulin Langmuir-Blodgett (LB) films transferred before the enzyme action were flat, while at the end of hydrolysis, roughness of films has increased and the appearance of 3D structures was observed.
authors.affiliation: Sofia University, Faculty of Chemistry, Department of Physical Chemistry, Lab Biophysical Chemistry, 1, James Bourchier Ave., Sofia 1164, Bulgaria. kbalashev@chem.uni-sofia.bg
authors.forename: K
authors.initials: K
authors.lastname: Balashev
authors.forename: Tz
authors.initials: T
authors.lastname: Ivanova
authors.forename: K
authors.initials: K
authors.lastname: Mircheva
authors.forename: I
authors.initials: I
authors.lastname: Panaiotov
mesh_terms: D000388:Air; D000818:Animals; D002417:Cattle; D004789:Enzyme Activation; D006868:Hydrolysis; D007328:Insulin / Q000737:chemistry*; D007700:Kinetics; D008567:Membranes, Artificial; D018625:Microscopy, Atomic Force; D011312:Pressure; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D013499:Surface Properties; D014867:Water / Q000737:chemistry
chemical_list: D007328:Insulin; D008567:Membranes, Artificial; D014867:Water; D012697:Serine Endopeptidases; C032923:microbial serine proteinases