The effect of pH on the kinetics of iron release from human transferrin

The rate of iron release from the N-terminal and C-terminal monoferrictransferrins (Fe_N-transferrin and Fe_C-transferrin, respectively) has been studied at 37°C over the pH range 3.5–10.6 using EDTA as the accepting chelate. Fe_N-transferrin is the more facile except above pH 8.2. Plots of log₁₀k_obs against pH showed a deviation for both monoferrictransferrins between pH 5.6 and 6.0 and studies above and below this transition point indicated that iron release occures by different mechanisms. At low pH (< 5.6) the rate of release from Fe_N-transferrin is independent of the presence of EDTA or NaClO₄, whereas Fe_c-transferrin shows a small but significant increase with increasing EDTA concentration. Rapid protonation of both monoferrictransferrins is followed by relatively slow release of Fe³⁺ which is subsequently chelated by EDTA. The slower release from Fe_c-transferrin is probably due to its greater binding strength for iron and the greater conformational stability of the C-terminal domain. Above pH 6.0 iron release from both monoferrictransferrins increases as the concentration of EDTA is increased. Direct attacks of EDTA probably occurs giving Fe-transferrin (HCO₃). EDTA as a transition state or intermediate. The factors which may lead to the observed pH dependence of the rate include (i) protonation of groups directly bound to the iron, (ii) conformers which differ in degree of protonation and (iii) the degree of protonation of the attacking chelating agent. It is suggested that an increase in conformational fluctuations as the pH is lowered may play a very important role. Studies with differrictransferrin at pH 4.53 and 7.40 showed that when iron is released to EDTA the rate is independent of the occupancy of the other site; that is, the two sites are exhibiting non-co-operativity.