Glutamic acid 235 and tryptophan residues are important for EndoS activity. Panel A, homology model of the amino-terminal part of EndoS with glutamic acid 235 (red) and the tryptophan residues (blue) are highlighted. Panel B, dilutions of rEndoS or rEndoS(E235Q) incubated with 1 μg human IgG for 1 hour at 37°C. IgG-glycan hydrolysis by EndoS was analyzed by SDS-PAGE and LCA-blot. Panel C, human IgG incubated with buffer, rEndoS modified with NEM, NBS, IAA, or unmodified (Buffer). The proteins were separated by SDS-PAGE, stained or analyzed for LCA reactivity in blot.