FIG. 4.

Formation of NE-Tf complexes. (a and b) Sephadex G-25 elution profiles of 100 μg of [⁵⁵Fe]Tf in the absence of NE and following incubation with 50 μM NE, respectively. The radioactivity in 30-μl aliquots of 0.5-ml column fractions was determined by scintillation counting. (c) Coomassie-stained urea-polyacrylamide gel showing protein profiles of peak material from the Sephadex G-25 columns shown in panels a and b. Mobilities of marker isoforms of Tf are as described in the legend to Fig. 3. (d) Sephadex G-25 elution profile of 100 μg of iron-saturated Tf following incubation with [³H]NE. The radioactivity in 30-μl aliquots of 0.5-ml column fractions was determined by scintillation counting; the label associated with the Tf peak represents approximately 8% of the total radioactivity. (e) Viable counts of E. coli strain E2348/69 after 18 h of incubation from an inoculum of 10² CFU/ml in unsupplemented serum-SAPI medium (control) or in serum-SAPI medium supplemented with NE complexes of (i) the Tf isoform mixture used as markers in panel c above, (ii) Apo-Tf, (iii) iron-bound Lf, or (iv) BSA. (f) Binding of [³H]NE, expressed as ³H cpm per 30 μg of protein, to (i) partially iron-saturated Tf, (ii) Apo-Tf, (iii) iron-bound Lf, or (iv) BSA; the values shown are the means of triplicate assays.