Medline/PubMed Citation | Related Articles in PubMed | Download to Citation Manager

Biotechnology and Applied Biochemistry (2002) 36, (21–31) (Printed in Great Britain)

PDF

Legacy HTML

Modification of a reactive cysteine explains differences between rasburicase and Uricozyme^®, a natural Aspergillus flavus uricase

Alain Bayol*¹, Joël Capdevielle†, Pascal Malazzi*, Armelle Buzy†, Marie Claude Bonnet*, Nathalie Colloc'h‡, Jean-Paul Mornon§, Denis Loyaux† and Pascual Ferrara†

*Analysis and Quality Control Unit, Sanofi-Synthelabo Recherche, Innopole, Voie no. 1, BP 137, 31676 Labège Cédex, France, †Protein Structure Group, Molecular and Functional Department, Sanofi-Synthelabo Recherche, Innopole, Voie no. 1 BP 137, 31676 Labège Cédex, France, ‡Université de Caen, CNRS UMR6551, BP 14074, Boulevard Becquerel, 14074 Caen Cédex, France, and §LMCP, UP6 and UP7, CNRS UMR 7590, case 115, 4 Place Jussieu, 75252 Paris Cédex 05, France

Key words: cysteine adduct, mass spectrometry, reverse-phase chromatography, zymogram.

Abbreviations used: DTT, dithiothreitol; ESI, electrospray ionization; IEF, isoelectrofocusing; MALDI, matrix-assisted laser-desorption ionization; QTOF, quadripole time-of-flight; Th, Thompson unit; C_mod, cysteine modified by the addition of a free cysteine residue.

¹To whom correspondence should be addressed (e-mail alain.bayol@sanofi-synthelabo.com).

Urate oxidase is used in humans for the control of uric acid in patients receiving chemotherapy. Rasburicase (Fasturtec^®/Elitek^®), a recombinant urate oxidase expressed in Saccharomyces cerevisiae, was compared with Uricozyme^®, the natural enzyme produced by Aspergillus flavus. Rasburicase has a higher purity as demonstrated by SDS/PAGE and chromatographic analysis and a better specific activity. The differences observed for Uricozyme^® are likely attributable to the previously used purification process, which modifies the enzyme. The production process of rasburicase, on the other hand, preserves the structure of the molecule. MS analysis shows that Uricozyme^® contains a cysteine adduct on Cys¹⁰³. In the crystal structure, the sulphur atom of the cysteine residue in position 103 is orientated to the external surface of the tetramer, whereas the sulphur atom of two other cysteine residues (Cys³⁵ and Cys²⁹⁰) is orientated to the centre of the canal formed by the tetramer. The same adduct is produced by simple incubation of the rasburicase with cysteine.

Received 10 October 2001/26 March 2002; accepted 11 April 2002

Portland Press Ltd © 2002