Effects of heavy metals on iron and zinc chelation during hepatic heme biosynthesis

This thesis reports the development of two new assay methods for Ferrochelatase. The first allows fluorimetric measurement of both Deuteroporphyrin (DP) and the Zinc chelate after stop-time assays. This method allows the simultaneous determination of DP remaining and the Zn-chelate formed, since it was found that the emission wavelengths of DP and ZnDP differ by 41 nm. When the excitation wavelengths chosen for each component are also separated by 41 nm, complete resolution is possible in a suitably diluted mixture.The second method allows continuous spectroscopic measurement of ZnDP formation by using the extinction coefficient determined from visible absorption spectra.A preliminary investigation into the applicability of Magnetic Circular Dichroism Spectroscopy revealed that this instrument may be used to quantify a mixture of ZnPP and Heme in a sample such as blood, with minimal handling procedures.The activity of solubilized rat liver mitochondrial Iron-chelatase was determined using a variety of reducing agents. Also, the effect of the Cytochrome Oxidase inhibitors CO and CN on particulate Fe-chelatase with Fe3+ and Fe2+ as substrates, revealed several aspects of this activity's requirement for reducing conditions.The non-enzymic chelation of Zinc with DP is qualitatively determined, and a hypothesis is presented to account for the observed results. The effects of the metal ions; Pb2+, Hg2+, Cd2+, Cu2+, As3+, and Zn2+ or Fe2+ on Iron-chelation and Zinc-chelation (respectively) are reported, as are the comparative effects of pCMB.It is shown, for the first time, that Lead may directly enhance the Zinc-chelatase activity of mitochondrial membrane preparations, while at the same time inhibiting Iron chelation in vitro. This finding may explain the clinical observation of ZnPP accumulation during Lead poisoning without requiring lead to secondarily affect putative iron transport processes. The general initial velocity rate equation for bi-substrate reactions was used to analyze the control plots. It was found that the binding of one substrate does not affect the binding of the other (Kia=Ka), so that apparent Km values could be obtained directly from Eadie-Scatchard plots:For Iron chelation; KFe - 16 ±11 uM, KDp = 17 ±11 uM.For Zinc chelation; KZn = 11 ±2 uM, KDP = 4 ±1 uM.This also demonstrates the kinetic mechanism is sequential.The kinetic pattern of lead inhibition indicated lead competes with iron binding but does not affect DP binding. Both the Log plot for Fe-chelation inhibition by lead and the parabolic secondary plot for the kinetic analysis indicated that either there are at least two binding sites for lead, both of which are sufficient to exclude iron from binding, or Fe-chelation activity is due to two or more isozymes with differing Vm and Km values.On the other hand lead activation of Zn-chelatase activity was non-competitive in both substrates, with a hyperbolic secondary plot. Three possible explanations are put forward . Lead binds to a site remote from both substrate binding sites so as to increase the productive breakdown of ternary complex. Secondly, Pb again does not affect substrate binding but interacts with two or more isomers with varying Vm and Km values. Thirdly, the activation may be a non-enzymic phenomenon.The implications of the observed non-linear reciprocal plot over a wider substrate concentration range are discussed.