title: Comparison of modification of a bacterial uricase with N-hydroxysuccinimide esters of succinate and carbonate of monomethoxyl poly(ethylene glycol).
abstract: Uricase after modification with monomethoxy poly(ethylene glycol) (mPEG) is currently the sole agent to treat refractory gout. For formulating Bacillus fastidious uricase, succinimidyl carbonate of mPEG-5000 (SC-mPEG5k) and succinimidyl succinate of mPEG-5000 (SS-mPEG5k) were compared. SC-mPEG5k possessed higher purity, comparable reaction rate constant with glycine but lower hydrolysis rate, and stronger effectiveness to modify amino groups. The uricase possessed two types of amino groups bearing a 25-fold difference in reactivity with SC-mPEG5k or SS-mPEG5k at pH 9.2. Oxonate and xanthine concentration-dependently protected the bacterial uricase from inactivation during PEGylation. With SC-mPEG5k at a molar ratio of 200 to uricase subunits and oxonate of 50 µM, the PEGylated uricase (1) retained about 73% of the original activity, (2) displayed about 10% reactivity to rabbit anti-sera recognizing the native uricase, (3) elicited IgG in rats accounting for about 5% of that by the native uricase, (4) exhibited circulation half-life time of about 25 H in cock plasma in vivo, and (5) concurrently maintained uric acid at lowered levels for over 20 H. Hence, PEGylation with SC-mPEG under the protection of a competitive inhibitor was a practical approach to formulation of the bacterial uricase; protection of enzymes by competitive inhibitors during PEGylation may have universal significance.
authors: Unit for Analytical Probes and Protein Biotechnology, Key Laboratory of Clinical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, Chongqing, People's Republic of China.
authors: Chun
authors: C
authors: Zhang
authors: Xiaolan
authors: X
authors: Yang
authors: Ang
authors: A
authors: Gao
authors: Xiaolei
authors: X
authors: Hu
authors: Jun
authors: J
authors: Pu
authors: Hongbo
authors: H
authors: Liu
authors: Juan
authors: J
authors: Feng
authors: Juan
authors: J
authors: Liao
authors: Yuanli
authors: Y
authors: Li
authors: Fei
authors: F
authors: Liao
mesh_terms: D000818:Animals; D001407:Bacillus / Q000737:chemistry* / Q000201:enzymology; D002254:Carbonates / Q000737:chemistry / Q000494:pharmacology; D002626:Chemistry, Pharmaceutical; D004952:Esters / Q000737:chemistry / Q000494:pharmacology; D006073:Gout / Q000188:drug therapy* / Q000473:pathology; D006801:Humans; D011092:Polyethylene Glycols / Q000737:chemistry / Q000494:pharmacology; D011817:Rabbits; D051381:Rats; D019802:Succinic Acid / Q000737:chemistry / Q000494:pharmacology; D013388:Succinimides / Q000737:chemistry* / Q000494:pharmacology; D014503:Urate Oxidase / Q000737:chemistry* / Q000627:therapeutic use
chemical_list: D002254:Carbonates; D004952:Esters; D013388:Succinimides; D011092:Polyethylene Glycols; C028210:monomethoxypolyethylene glycol; D019802:Succinic Acid; D014503:Urate Oxidase; C001426:N-hydroxysuccinimide
keywords: PEGylation; competitive inhibitor; protection; succinimidyl carbonate of mPEG; uricase