Figure 8.

Iron-binding cofactor in urine. (A) Buffer was mixed with ⁵⁵Fe (No protein, light blue) and with apo-Ngal (dark blue), apo-Ngal plus siderophore (yellow), or apo-Ngal plus siderophore plus unlabeled iron (pink). The samples were then washed 3 times on a 10-kDa filter, and small aliquots were measured for retention of radioactivity (Washes 1–3). After 48 hours at 4°C the samples were washed again (Wash 4 + 48 h). Note the retention of ⁵⁵Fe by apo-Ngal plus siderophore but not by apo-Ngal alone or apo-Ngal ligated by the iron-saturated siderophore, which demonstrates that an unsaturated siderophore is required for retention of ⁵⁵Fe by Ngal. ⁵⁵Fe binding to apo-Ngal plus siderophore was stable for 48 hours. (B) Urine (<3,000 Da) was mixed with ⁵⁵Fe and with apo-Ngal, as indicated, and then washed 3 times on a 10-kDa filter. While urine (<3,000 Da) or apo-Ngal in buffer did not retain ⁵⁵Fe on a 10-kDa filter, apo-Ngal plus urine retained ⁵⁵Fe. ⁵⁵Fe retention was blocked by the addition of excess iron citrate (Fe) or of iron-saturated enterochelin (Sid:Fe).