Modified high amylose starch for immobilization of uricase for therapeutic application.
Urate oxidase (uricase) was immobilized on carboxymethyl high amylose starch cross-linked 35 (CM-HASCL-35), on aminoethyl high amylose starch cross-linked 35, as well as on commercial supports, CNBr-activated Sepharose and diaminodipropylamine agarose. The N -ethyl-5-phenylisoxazolium-3'-sulphonate (Woodward reagent K) gave a high binding but totally inhibited the enzyme activity. Best results were obtained with CM-HASCL-35 using 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide as a coupling agent. The immobilized enzyme retained 88% of its initial limit rate [V (max)(app)=16 EU/mg for immobilized uricase versus V (max)=18 EU/mg for the free enzyme], with an apparent decrease of affinity for urate substrate [K (m)(app)=0.17 mM versus K (m)=0.03 mM for the free enzyme]. The coupling yield was 60% and the modified uricase was found more resistant to proteolysis than the free enzyme. The immobilized uricase retained 25% of its initial activity after 60 min in pancreatic proteolysis medium (pancreatin), whereas the free enzyme retained only 5% of its initial activity. The best immobilization yield was obtained with the polymeric support based on CM-HASCL-35 (53%), which gave better results than commercial supports based on agarose.
Mulhbacher
Jérome
J
Department of Chemistry and Biochemistry, Université du Québec à Montréal, C.P. 8888, Succ. A, Montréal, Québec, Canada H3C 3P8.
McGeeney
Kevin
K
Ispas-Szabo
Pompilia
P
Lenaerts
Vincent
V
Mateescu
Mircea-Alexandru
MA
D000688:Amylose / Q000737:chemistry*; D016503:Drug Delivery Systems / Q000379:methods*; D004789:Enzyme Activation; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry; D005079:Excipients / Q000737:chemistry*; D006073:Gout / Q000188:drug therapy; D006801:Humans; D006863:Hydrogen-Ion Concentration; D012685:Sepharose / Q000737:chemistry; D012995:Solubility; D013213:Starch / Q000737:chemistry*; D013379:Substrate Specificity; D014503:Urate Oxidase / Q000737:chemistry* / Q000627:therapeutic use
D004800:Enzymes, Immobilized; D005079:Excipients; D013213:Starch; D000688:Amylose; D012685:Sepharose; D014503:Urate Oxidase