Premium
Location of crosslinks in chemically stabilized horseradish peroxidase: Implications for design of crosslinks
Author(s) -
O'Brien Anne Marie,
Ó'Fágáin Ciarán,
Nielsen Per F.,
Welinder Karen G.
Publication year - 2001
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.1194
Subject(s) - horseradish peroxidase , chemistry , lysine , bifunctional , ethylene glycol , peptide , peroxidase , fragmentation (computing) , stereochemistry , combinatorial chemistry , biochemistry , enzyme , organic chemistry , amino acid , catalysis , biology , ecology
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.