Characterisation of chlorophyll oxidation mediated by peroxidative activity in olives (Olea europaea L.) cv. Hojiblanca

The oxidation of chlorophyll a (chl a) catalysed by peroxidase (POD) from mesocarp of the olive fruit (Olea europaea L., cv Hojiblanca) in the presence of H2O2 and 2,4-dichlorophenol (2,4-DCP), is characterised via the individualised quantification of the products of the enzymatic reaction using a new methodology of HPLC–UV spectrometry. This innovation has allowed the discovery that, in addition to 132 OH chl a and 151 OH lactone chl a, which are the first products of POD on chl a, the reaction process sequentially creates another series of oxidised chlorophyll derivatives which have not been previously described. Their origins have been linked to POD activity in the presence of 2,4-DCP. Likewise, a study of the effect of the concentration of the various cosubstrates on the POD reaction rate demonstrated that the correct establishment of the relative concentrations of the same ([H2O2]/[2,4-DCP]/[Chl] = 1:3:0.02) is crucial to explaining inhibition effects by substrates and carrying out optimum measurements. Therefore, new essential parameters for the determination of POD activity on a chlorophyll substrate are established.