Biochemical and biopharmaceutical properties of PEGylated uricase.
PEGylation is a successful strategy for improving the biochemical and biopharmaceutical properties of proteins and peptides through the covalent attachment of polyethylene glycol chains. In this work, purified recombinant uricase from Candida sp. (UC-r) was modified by PEGylation with metoxypolyethilenoglycol-p-nitrophenyl-carbonate (mPEG-pNP) and metoxypolyethyleneglycol-4,6-dichloro-s-triazine (mPEG-CN). The UC-r-mPEG-pNP and UC-r-mPEG-CN conjugates retained 87% and 75% enzyme activity respectively. The K(M) values obtained 2.7x10(-5) M (mPEG-pNP) or 3.0x10(-5) M (mPEG-CN) for the conjugates as compared to 5.4x10(-5) M for the native UC-r, suggesting enhancement in the substrate affinity of the enzyme attached. The effects of pH and temperature on PEGylated UC-r indicated that the conjugates were more active at close physiological pH and were stable up to 70 degrees C. Spectroscopic study performed by circular dichroism at 20 degrees C and 50 degrees C did not show any relevant difference in protein structure between native and PEGylated UC-r. In rabbit and Balb/c mice, the native UC-r elicited an intense immune response being highly immunogenic. On the other hand, the PEGylated UC-r when injected chronically in mice did not induce any detectable antibody response. This indicates sufficient reduction of the immunogenicity this enzyme by mPEG-pNP or mPEG-CN conjugation, making it suitable for a possible therapeutical use.
Freitas
Débora da Silva
Dda S
Department of Biochemical and Pharmaceutical Technology, Pharmaceutical Sciences School, University of São Paulo, Av. Prof. Lineu Prestes, Bloco 16, CEP 05508-000, São Paulo, SP, Brazil. debyrp@usp.b
Spencer
Patrick Jack
PJ
Vassão
Ruth Camargo
RC
Abrahão-Neto
José
J
D000818:Animals; D002175:Candida / Q000201:enzymology*; D002942:Circular Dichroism; D004337:Drug Carriers / Q000737:chemistry*; D004355:Drug Stability; D006863:Hydrogen-Ion Concentration; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D011092:Polyethylene Glycols / Q000737:chemistry*; D011817:Rabbits; D011994:Recombinant Proteins; D013696:Temperature; D014503:Urate Oxidase / Q000737:chemistry* / Q000276:immunology
D004337:Drug Carriers; D011994:Recombinant Proteins; D011092:Polyethylene Glycols; C028210:monomethoxypolyethylene glycol; D014503:Urate Oxidase