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

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.