Degradation of Chlorophylls: Purification and properties of a Mg-releasing protein from Chenopodium album

Abstract

In the early stage of the degradation of chlorophyll, the modification of the side chains of the macro and isocyclic rings of chlorophyll occurs at least three steps of reactions. An involvement of Mg-dechelatase have been suggested at the second step of Mg-releasing reaction, however, its presence is unclear. Previously, we reported that there was small molecular weight substance which chelates Mg of chlorophyllide. However, it is uncertain whether Mg-dechelatase itself or not. In the present study, we reexamine Mg-dechelating reaction with an assay method using chlorophyllin (Mg-chlorin) as a substrate. The results revealed a presence of protein which has Mg-releasing ability. Here, we report the purification and characterization of Mg-releasing protein. Acetone powder from Chenopodium album was used as the starting material. For assay of Mg-releasing protein, a reaction product, pheophorbin (Mg-free chlorin), was determined from increase in absorbance at 692 nm. Mg-releasing protein was purified 28-fold by ammonium sulfate fractionation (30-75% saturation), DEAE, Butyl and HW-55 chromatography. pH optimum of the purified preparation was 7.5. Km value for chlorophyllin was 95.1 nM. Molecular weight estimated by gel filtration was about 20 k. The enzyme reaction was strongly inhibited by sodium ascorbate, propyl gallate and hydroquinone. The Mg-releasing protein had no activity for chlorophyllide a, a true substrate. It is known that horseradish peroxydase shows similar Mg-releasing activity, however, it had no activity against chlorophyllide a as well. We also report the properties of Mg-releasing protein in comparison with those of metal-chelating substance.