In vitro studies on ethylene-enhanced chlorophyll degrading peroxidase and its reaction products in banana (Musa sapientum L.) Fruits

Abstract

Ethylene enhanced degreening of intact banana (Musa sapientum L.) peel in the dark. Enzymes from ethylene-treated fruits of banana were isolated which catalyzed the degradation of chlorophyll a in the presence of H2O2 and 2,4-dichlorophenol. Its pH optima are 5.2, 5.8, and 6.4 with potassium phosphate buffer. Reaction was strongly inhibited by hydroquinone, Tiron, Mn2+, L-sodium ascorbate, n-propyl gallate, salicylhydroxamic acid, KCN, and NaN3 and weakly by 2.2'-bipyridyl and 1,10-phenanthroline. The participation of Fe2+ ⇆ Fe3+ and O2/- in the reaction is discussed. Chlorophyll degrading peroxidases are clearly distinct from guaiacol peroxidase in its inhibition properties upon NaN3. Linearity between the rate of the its degrading activity and protein concentration was obtained over the range 0.04 ~ 0.33 mg protein per 3.0 ml of the reaction mixture. Km for chlorophyll a and H2O2 is approximately 16.5 μM and 20.44 μM, respectively. The possible in vivo participation of this in vitro system is briefly discussed. Visible adsorption spectrum of the reaction mixture during chlorophyll a degradation indicated a shift of the red band near 670 nm to a shorter wavelength and a shift of the Soret band near 430 nm to a longer wavelength. Difference spectrum of the reaction mixtures showed a peak near 450 nm. The yellow peak indicates the presence of open-ring catabolites and the lack of the Soret and red bands. The appearance of a fluorescent chlorophyll catabolite (Ex 350nm, Em 465 nm) was recognized.