Table 2. Millimolar absorption coefficients (ε) at 280 nm for full length hTF and oTF.

All samples were assayed in 100 mM HEPES, pH 7.4 (iron-bound) or 100 mM Acetate, pH 4.0 and 4 mM EDTA (Apo).

Protein	Calculated ε280 (Apo)a	Experimental ε280 (Apo)b	Experimental ε280 (Iron)b	% Increase due to ironc	% Difference Calc/Expc
Diferric hTF	85.1	84.0 ± 0.2	103.9 ± 0.2	23.7	−1.3
Monoferric-N hTFd	82.1	81.4 ± 0.3	92.5 ± 0.3	13.6	−0.9
Monoferric-C hTFe	82.1	81.5 ± 0.2	92.1 ± 0.2	13.0	−0.7
Apo hTFf	79.2	80.1 ± 0.4	-	-	1.1
Diferric oTF	88.2	87.9 ± 0.1	109.3 ± 0.2	24.3	−0.3

^aCalculated from Eq. 2.

^bValues are means ± STD of at least three determinations.

^cPercent increase and difference are calculated as in Table 1.

^dRecombinant non-glycosylated His tagged hTF with Y426F and Y517F mutations inhibiting its ability to coordinate iron in the C-lobe.

^eRecombinant non-glycosylated His tagged hTF with Y95F and Y188F mutations inhibiting its ability to coordinate iron in the N-lobe.

^fThis is non-glycosylated authentic recombinant His tagged apo-hTF mutant (Y95F/Y188F/Y426F/Y517F).