Purification and Properties of Chlorophyllide a Breaching Enzyme (Chlorophyllide a Peroxidase) from Citrus unshiu Fruits

Abstract

A chlorophyllide a breaching enzyme extracted from degreening Citrus unshiu fruits was purified some 1, 000-fold and characterized.
1. The enzyme catalyses chlorophyllide a degradation in the presence of H₂O₂ and the phenols, 2, 4-dichlorophenol or p-coumaric acid. It behaves as a protein with a MW of 42, 000 on Sephacryl S-200 gel filtration but it does not give a single band on polyacrylamide gel electrophoresis (PAGE), SDS-PAGE, and upon staining with guaiacol in the presence of H₂O₂. The Michaelis constant, Km, for chlorophyllide a was 13.2 μM.
2. The enzyme exhibited IAA oxidase activity in the presence of Mn⁺⁺; it was inhibited by hydroquinone, Tiron, potassium cyanide (KCN), diethyldithiocarbamate (DIECA) but not by iodoacetate, p-chloromercuribenzoate (PCMB), N-ethylmaleimide, histidine, D-mannose, dimethylfuran, triethylenediamine, ethylenediaminetetraacetic acid (EDTA), NaN₃ and thiourea.
The possible in vivo participation of this in vitro enzyme system as a H₂O₂-scavenging system in the dark is discussed.