TABLE 3.

In vitro ENGase activity assay using POSs with various glycan structures

^aGlycan structures were determined by the elution positions of both size-fractionation and dual-gradient reversed-phase HPLC and comparison with authentic standard glycans (25). To verify the position of Gn1-type FNGs under the HPLC conditions used, the reaction products were analyzed in parallel with POSs that were digested with Endo-H (for Glc₃Man₉GlcNAc₂-P and Man₉GlcNAc₂-P) or Endo-M (Man₅GlcNAc₂-P and Man₃GlcNAc₂-P).

^bThe amounts of reaction products were estimated from peak area of PA-Glc₆ in size-fractionation HPLC. The hydrolytic rates were estimated based on the amounts of Gn1-type FNGs at 1 h (Glc₃Man₉GlcNAc₁, Man₉GlcNAc₁, and Man₅GlcNAc₁) and 2 h (Man₃GlcNAc₁) after incubation.

^cHydrolytic rates of Man₉GlcNAc₂-P were set to 1.0.