Secreted IL4I1 presents a phenylalanine oxidase activity over a wide pH range. (A) Culture medium (60 μL) or 100 μg cell lysate from hIL4I1-HEK induced with tetracycline was used to measure LAAO activity in a colorimetric assay (“Materials and methods”) against l-phenylalanine. Results are expressed as nanomoles of H₂O₂ produced in a 2-hour reaction. (B) Medium (60 μL) from wild-type human (■), E481A mutant (□), and mouse IL4I1 (▩) expressing HEK cells were used to measure LAAO activity as in panel A. (C) hIL4I1-HEK cell extracts (100 μg total protein) were tested at pH 5 for LAAO activity against l-amino acids and d-leucine (one letter nomenclature) as in panel A. (Inset) LAAO activity of 100 μg total protein was measured against phenylalanine at different pHs. Mean ± SD from 3 (A-B) or 2 (C) independent experiments is shown.