Red clover polyphenol oxidase: Activation, activity and efficacy under grazing

This study investigated red clover polyphenol oxidase (PPO), its: activity, activation from latent into active form, and whether grazing at different levels of herbage maturity could result in sufficient PPO activation to protect proteins and glycerol-based lipid in the rumen. Wild-type red clover with high PPO activity and a mutant red clover with low PPO activity were used in five experiments. Firstly to investigate activation of the latent red clover PPO enzyme either through: protease, substrate (ortho-diphenols), cell damage or chemically (sodium dodecylsulphate detergent) induced mediation. Secondly PPO's activity at a range of pHs were determined to show how activation method altered the enzymes activity. Finally whether grazing could activate latent PPO and ultimately result in protected protein and glycerol-based lipid in the rumen was investigated. Incubation of extracted red clover PPO enzyme with a range of ortho-diphenols in the presence of oxygen resulted in rapid conversion of the latent to the active form of PPO. This conversion was blocked by the addition of ascorbic acid suggesting that this mechanism requires formation of ortho-quinones. Cell damage was highly correlated with activation of latent PPO and the formation of protein-bound phenols due to mixing of the chloroplastic enzyme with the vacuolar substrate (ortho-diphenols). The potential for PPO activity to occur during grazing was investigated by measuring the oxygen concentration in red clover boluses and within the rumen and PPO's effect on protein and glycerol-based lipid within red clover boluses. There was a rapid depletion of oxygen from the rumen, limiting the potential for PPO activation and activity to occur to the period of mastication. PPO was shown to be activated in both wild-type and mutant red clover boluses, however, factors such as fibre content and maturity of the red clover herbage (PPO concentration) altered the efficacy of PPO to produce phenol-bound protein. As a result greater protection of glycerol-based lipid in this study was achieved with a prior conditioning (wilting) of the red clover.