Location of crosslinks in chemically stabilized horseradish peroxidase: Implications for design of crosslinks

  1. Anne Marie O'Brien1,
  2. Ciarán Ó'Fágáin1,*,
  3. Per F. Nielsen2,
  4. Karen G. Welinder3,†

Article first published online: 13 NOV 2001

DOI: 10.1002/bit.1194

Issue

Biotechnology and Bioengineering

Biotechnology and Bioengineering

Volume 76, Issue 4, pages 277–284, December 2001

Additional Information

How to Cite

O'Brien, A. M., Ó'Fágáin, C., Nielsen, P. F. and Welinder, K. G. (2001), Location of crosslinks in chemically stabilized horseradish peroxidase: Implications for design of crosslinks. Biotechnol. Bioeng., 76: 277–284. doi: 10.1002/bit.1194

Author Information

  1. 1

    School of Biotechnology, Dublin City University, Dublin 9, Republic of Ireland; telephone: +353-1-700-5288; fax: +353-1-700-5412

  2. 2

    Novo Nordisk A/S, Novo Alle, Bagsværd, Denmark

  3. 3

    Department of Protein Chemistry, Institute of Molecular Biology, University of Copenhagen, Copenhagen, Denmark

  1. †

    Present address: Department of Life Science, Aalborg University, Aalborg, Denmark

*School of Biotechnology, Dublin City University, Dublin 9, Republic of Ireland; telephone: +353-1-700-5288; fax: +353-1-700-5412

Publication History

  1. Issue published online: 13 NOV 2001
  2. Article first published online: 13 NOV 2001
  3. Manuscript Accepted: 9 FEB 2001
  4. Manuscript Received: 25 MAY 2000

Funded by

  • European Commission Fourth Framework Biotechnology Programme, “Designer Peroxidases”. Grant Number: BIO4-CT97-2031

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Keywords:

  • peroxidase;
  • stabilized;
  • crosslinked;
  • peptide mapping;
  • mass spectrometry;
  • modified lysines;
  • surface accessibility

Abstract

The bifunctional compound, ethylene-glycol bis(N-hydroxysuccinimidylsuccinate) (EGNHS), stabilizes horseradish peroxidase C (HRP) by reaction with the enzyme's lysine residues. In this study we compare native and modified HRP by proteolytic fragmentation, peptide sequencing, and mass spectroscopy, and identify the sites of modification. Most significantly, EGNHS is shown to form a crosslink between Lys232 and Lys241 of HRP and modifies Lys174 without formation of a crosslink. These findings are in agreement with the lysine side-chain reactivities predicted from the surface accessibility of the amino groups, and the maximal span of 16 Å of the EGNHS crosslinker. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 76: 277–284, 2001.

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