Studies on the catalytic behaviour of a cholinesterase-like abzyme in an AOT microemulsion system.
The hydrolytic activity of a monoclonal catalytic antibody (9A8) (abzyme) with acetylcholinesterase-like activity was investigated in water-in-oil (w/o) microemulsions (reverse micelles) based on sodium bis-2-(ethylhexyl)sulfosuccinate (AOT) in isooctane, using p- and o-nitrophenylacetate (p-and o-NPA) as substrates. The dependence of the abzyme hydrolytic activity on the molar ratio of water to surfactant (w(o)) showed a bell-shaped curve, presenting a maximum at w(o)=11.1. An increase of the AOT concentration at constant w(o), resulted in a decrease of the catalytic activity suggesting a possible inhibition effect of the surfactant. The incorporation of the abzyme into the reverse micelle system caused a blue shift of the fluorescence emission maximum by a magnitude of 7-10 nm depending on the w(o) value. This result indicates that the antibody molecule, or a large part of it, is located in the aqueous microphase of the system. Kinetic studies showed that the hydrolysis of p-and o-NPA in microemulsion system as well as in aqueous solution follows Michaelis-Menten kinetics. The catalytic efficiency (k(cat)/K(m)) in w/o microemulsion was significant lower than in aqueous solution.
Franqueville
Eric
E
Biotechnology Laboratory, Division IV, Chemical Engineering Department, National Technical University of Athens, Zografou campus, 15700 Athens, Greece.
Stamatis
Haralambos
H
Loutrari
Heleni
H
Friboulet
Alain
A
Kolisis
Fragiskos
F
D000818:Animals; D017151:Antibodies, Catalytic / Q000096:biosynthesis / Q000737:chemistry*; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry*; D002384:Catalysis; D002802:Cholinesterases / Q000737:chemistry; D004143:Dioctyl Sulfosuccinic Acid / Q000737:chemistry*; D004655:Emulsions; D004795:Enzyme Stability; D004800:Enzymes, Immobilized; D006868:Hydrolysis; D007075:Immunoglobulin M / Q000737:chemistry*; D051379:Mice; D008823:Micelles; D009578:Nitrobenzenes / Q000737:chemistry; D009795:Octanes / Q000737:chemistry*; D009821:Oils / Q000737:chemistry; D010648:Phenylacetates / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012995:Solubility; D013379:Substrate Specificity; D013501:Surface-Active Agents; D014867:Water / Q000737:chemistry / Q000378:metabolism*
D017151:Antibodies, Catalytic; D000911:Antibodies, Monoclonal; D004655:Emulsions; D004800:Enzymes, Immobilized; D007075:Immunoglobulin M; D008823:Micelles; D009578:Nitrobenzenes; D009795:Octanes; D009821:Oils; D010648:Phenylacetates; D013501:Surface-Active Agents; D014867:Water; D004143:Dioctyl Sulfosuccinic Acid; C119971:2-nitrophenylacetic acid; D002802:Cholinesterases; C118008:4-nitrophenylacetic acid; C045798:2,2,4-trimethylpentane