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Immobilized purine nucleoside phosphorylase from Schistosoma mansoni for specific inhibition studies

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Abstract

The parasite Schistosoma mansoni (Sm) depends exclusively on the salvage pathway for its purine requirements. The enzyme purine nucleoside phosphorylase (PNP) is, therefore, a promising target for development of antischistosomal agents and an assay for screening of inhibitors. To enable this, immobilized SmPNP reactors were produced. By quantification of hypoxanthine by liquid chromatography, kinetic constants (K M) for the substrate inosine were determined for the free and immobilized enzyme as 110 ± 6.90 μmol L−1 and 164 ± 13.4 μmol L−1, respectively, indicating that immobilization did not affect enzyme activity. Furthermore, the enzyme retained 25 % of its activity after four months. Non-Michaelis kinetics for the phosphate substrate, and capacity for Pi-independent hydrolysis were also demonstrated, despite the low rate of enzymatic catalysis. Use of an SmPNP immobilized enzyme reactor (IMER) for inhibitor-screening assays was demonstrated with a small library of 9-deazaguanine analogues. The method had high selectivity and specificity compared with screening by use of the free enzyme by the Kalckar method, and furnished results without the need for verification of the absence of false positives.

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Acknowledgments

This work was funded by grants from the São Paulo State Research Foundation (FAPESP process 2008/04051-0). The authors also thank the National Council for Scientific and Technological Development (CNPq). The authors acknowledge also Dr Renata Krogh and Adriano D. Andricopulo, from Instituto de Física de São Carlos, Universidade de São Paulo, São Carlos, Brazil, for helpful discussion, for helping with expression and purification of SmPNP, and also, for furnishing the 9-deazaguanine analogues.

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Authors and Affiliations

  1. Departamento de Química, Universidade Federal de São Carlos, Cx Postal 676, São Carlos, 13565-905, São Paulo, Brazil

    Marcela Cristina de Moraes & Quezia B. Cass

  2. Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, 14040-901, São Paulo, Brazil

    Carmen L. Cardoso

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  1. Marcela Cristina de Moraes
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  2. Carmen L. Cardoso
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Correspondence to Quezia B. Cass.

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de Moraes, M.C., Cardoso, C.L. & Cass, Q.B. Immobilized purine nucleoside phosphorylase from Schistosoma mansoni for specific inhibition studies. Anal Bioanal Chem 405, 4871–4878 (2013). https://doi.org/10.1007/s00216-013-6872-7

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  • DOI: https://doi.org/10.1007/s00216-013-6872-7

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