The effect of pH on the kinetics of iron release from diferric ovotransferrin induced by pyrophosphate.

Pyrophosphate-induced iron release from diferric ovotransferrin follows biphasic kinetics in the pH range from 6.6 to 8.6 except at pH 8.0 where the kinetics become monophasic. The rates of formation of the four molecular species, Fe2OT, FeOTN, FeOTC, and ApoOT, were studied by urea gel electrophoresis and the four microscopic rate constants were calculated at various pH values. Below pH 8.0, these intrinsic rate constants for iron release from Fe2OT follow the order k2N greater than k1N greater than k2C greater than k1C. Each constant diminishes almost proportionally with an increase in pH with the faster rate constants being affected more by the fall in hydrogen ions than the slower ones. Around pH 8.0 the four rates are approximately equal, resulting in monophasic kinetics. However, the rate constants from the C-site become faster than that from the N-site at pH above 8.2. At low pH, there is a marked preference for iron to be released from the N-site rather than from the C-site and such preference becomes less distinct as pH increases. A rather weak positive cooperativity between the two sites is demonstrated in pH between 6.8 and 7.8. The ligand responsible for the transition from biphasic to monophasic kinetics at pH 8.0 is not known. It is possible that there are different anions such as [CO3(2-)] and [HCO3-] at the two iron-binding sites, which might explain the preferential rates of iron release from these sites during protonation.

Kinetics of pyrophosphate induced iron release from diferric ovotransferrin.

The kinetics of pyrophosphate-induced iron release from diferric ovotransferrin were studied spectrophotometrically at 37 degrees C in 0.1 M HEPES, pH 7.0. At high pyrophosphate concentrations, the kinetics are biphasic, indicating that the rates of iron release from the two, presumably noninteracting iron-binding sites of ovotransferrin are different. The pseudo-first-order rate constants for iron release from both the fast and slow sites exhibit a hyperbolic dependence on pyrophosphate concentrations. The data suggest that pyrophosphate forms complexes with the two iron-binding sites of ovotransferrin prior to iron removal. The stability constants of the complex formed with the fast site (Keqf) and slow site (Keqs) are 8.3 M-1 and 40.4 M-1, respectively. The first-order rate constants for the dissociation of ferric-pyrophosphate from the fast site (k2f) and the slow site (k2s) are 0.062 and 0.0044 min-1, respectively. Results from urea gel electrophoresis studies suggest that iron is released at a much faster rate from the N-terminal binding site of ovotransferrin. At high pyrophosphate concentration, only C-monoferric-ovotransferrin is detected during the course of iron release. At low pyrophosphate concentration, however, a detectable amount of N-monoferric-ovotransferrin is accumulated. This result is consistent with the kinetic finding that the site with a higher k2 (0.062 min-1) has a lower affinity toward pyrophosphate (Keq = 8.3 M-1) whereas the site with a lower k2 (0.0044 min-1) has a higher affinity for pyrophosphate (Keq = 40.4 M-1).