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Synthesis and Characterization of Polysialic Acid–Uricase Conjugates for the Treatment of Hyperuricemia

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Abstract

Uricase, an enzyme used for the treatment of hyperuricemia, is conjugated with polysialic acid (PSA) of average molecular weight of 10 kDa by reductive amination in presence of NaCNBH3 in order to improve its pharmacological properties. Polysialylation with 50-,100-,150- and 200-fold molar excess of PSA increased the percentage substitution of the free amino groups on enzyme surface (46, 66, 78 and 80 % respectively). The SDS-PAGE is used to visualize the conjugates with increased molecular weight and it retained almost 65 % of their initial specific activity after conjugation. The stability studies at physiological condition reveals improved stability and activity than the native enzyme. The apparent KM of the enzyme has increased slightly from 4.18 × 10−5 M to 5.46 × 10−5 M suggesting that the affinity of the substrate to the enzyme has not been altered to a higher extent. The conjugates, when probed against anti-uricase antibodies generated in rabbit, showed a clean decline in the affinity by 35 % and also have retained double the catalytic activity than that of the native enzyme after exposure to antiserum. The results suggest that uricase-PSA conjugates can be used as an alternative to the conventional synthetic polymer-enzyme conjugates.

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Conflict of Interest and Ethical Standards

There is no conflict of interest between the authors or any institutions in this project and there is no external funding involved.

Statement of Informed Consent

There are no human subjects involved in this study and any information on informed consent is not relevant.

Statement of Human and Animal Rights

This study was approved by Institutional Animal Ethics Committee of Kasturba Medical College, Mangalore, India, in IAEC meeting held on 14/06/2013 and has followed the national guide for the care and use of laboratory animals.

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Authors and Affiliations

  1. Department of Chemical Engineering, National Institute of Technology Karnataka, Srinivasanagar, Surathkal, Mangalore, 575025, Karnataka, India

    Ardra Punnappuzha & JagadeeshBabu PonnanEttiyappan

  2. Department of Pharmacology, Kasturba Medical College, Light House Hill Road, Hampankatta, Mangalore, 575001, Karnataka, India

    Reval Sekhar Nishith, Sanjay Hadigal & Preethi Ganapathi Pai

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  1. Ardra Punnappuzha
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  2. JagadeeshBabu PonnanEttiyappan
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Correspondence to JagadeeshBabu PonnanEttiyappan.

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Punnappuzha, A., PonnanEttiyappan, J., Nishith, R.S. et al. Synthesis and Characterization of Polysialic Acid–Uricase Conjugates for the Treatment of Hyperuricemia. Int J Pept Res Ther 20, 465–472 (2014). https://doi.org/10.1007/s10989-014-9411-2

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