FIGURE 5.

Identification of binding sites between NEU1 and PPCA by SPR-peptide scanning. A, purified NEU1 was covalently coupled to a SPR sensor chip. Binding of PPCA was measured by flowing purified wild-type (black plot) or mutant precursor PPCAs (red, green, and blue plots) at pH ranging from 4.5 to 7.5 through the reference and the NEU1-containing flow cells (A). B, SPR binding of a series of 88 synthetic PPCA peptides (15-mer with 10-residue overlap) was measured by flowing the peptides in sequence through the reference and protein-containing flow cells. C and D, SPR binding of a series of 72 synthetic NEU1 peptides to precursor PPCA (C), mature PPCA (D), and NEU1 (E). Data reported are the differences in SPR signal between the flow cells containing protein and the reference cells. The binding level was determined as the maximum signal in the binding phase. Peptides that gave binding values that were higher than the nonspecific background values are indicated.