HPLC was performed with a Waters Delta 600 pump, a Waters 600 controller, and a Waters 2996 photodiode array (PDA) detector with Empower 2 software (Waters Ltd., Mississauga, ON, Canada). Bovine submaxillary mucin Type I-S, 9-24 % bound sialic acids was purchased from Sigma Aldrich. Neuraminidases from Clostridium perfringens (NanI) and Arthrobacter ureafaciens (siaAU) were purchased from Sigma Aldrich. The human neuraminidase enzymes NEU2, NEU3, and NEU4 were expressed as fusion proteins with maltose binding protein, as previously reported. [47] [48] [49] NEU1 was overexpressed in HEK293E cells and used as crude cell lysate. 7 Specific activity of the neuraminidase enzymes were normalized in comparison to a standard curve with NanI using 4-methylumbelliferyl α-D-N-acetylneuraminic acid (4MU-NANA) as a substrate.
Unless otherwise stated, results reported are technical replicates, with preliminary independent replicates confirming the trends reported.
Unless otherwise stated, results reported are technical replicates, with preliminary independent replicates confirming the trends reported.