The Encapsulation of Squid Diisopropylphosphorofluoridate-Hydrolyzing Enzyme within Mouse Erythrocytes

This study describes the entrapment of squid-type diisopropylphosphorofluoridate-hydrolyzingenzyme (DFPase) within mouse red blood cells. These erythrocytesthereby gain the ability to rapidly hydrolyze alkylphosphatecholinesterase (ChE) inhibitors such as diisopropyl fluorophosphate(DFP). DFPase rapidly hydrolyzes DFP to diisopropyl phosphate.Resealed erythrocytes provide a stable carrier system that canpreserve the activity of encapsulated enzymes against otherwiserapid in vivo degradation; thus, ChE inhibitors can be degradedto relatively nontoxic metabolites by these erythrocyte carriers.Squid DFPase was purified from the hepatopancreas of Atlanticsquid and DFPase activity was determined by measuring changesin fluoride ion concentration using a fluoride ion selectiveelectrode. Mouse erythrocytes in suspension with excess squidDFPase were dialyzed against hypotonic buffer to allow the encapsulationof the enzyme to occur. Cells were then resealed by returningthe suspension to isosmotic with saline. Rate of DFP hydrolysisobserved with these cells was much greater than the rate ofnonenzymatic hydrolysis and was directly proportional to theamount of the erythrocyte suspension added to the assay solution.The rate of hydrolysis was first order in substrate. Erythrocytecontrols showed no endogenous DFPase activity. These resultssuggest that enzyme entrapment may be developed as a methodto prevent and antagonize organophosphate poisoning.