ARS | Publication request: Auto-Oxidation of Ortho-Diphenolic Substrate and Deactivation of Polyphenol Oxidases (Catecholase) During Wilting and Post Harvest Damage in Red Clover

Submitted to: Abstracts World Buiatrics Congress
Publication Type: Abstract Only
Publication Acceptance Date: August 2, 2010
Publication Date: N/A

Technical Abstract: Polyphenol oxidases (PPO) in red clover convert diphenolic substrate to highly reactive quinones which, through their reaction with proteins, increase the efficiency of N utilization and increase the proportion of beneficial polyunsaturated fatty acids in bovine products (meat and milk). Auto-oxidation of phenolic substrate to quinone has been previously reported, but little information is available on the degree of auto-oxidation in forage crops during conservation, with all activity being attributed to enzymatic oxidation through PPO. This study investigated the degree of oxidation in wild type red clover (PPO+) and red clover with the PPO1 gene silenced by genetic manipulation (PPO-) to determine: 1) the degree of auto-oxidation under two damage regimes (heavy and light), and 2) the temporal denaturation of PPO. PPO+ and PPO- red clover plants were grown under controlled conditions and harvested at six weeks regrowth. The material was passed through a garden shredder as the light damage (LD) and half frozen at -20 degrees C as the heavy damage (HD). Material was left at room temperature and sampled at regular intervals for determination of PPO enzyme activity (active and total), PPO activation, and formation of protein-bound phenol (PBP). For PPO+, HD-active PPO was higher (P<0.05) than PPO+ LD during the initial time points (0-0.5 h), whereas total PPO were comparable under both damage regimes. After 1 h, both total and active PPO for HD dropped to below LD and trailed off to negligible activity at 24 h. The LD-active PPO increased (P<0.05) up to 1 h before declining and stabilizing after 4 h. PPO- had no detectible activity throughout the experiment. PBP formation as a measure of oxidation was highest initially on the PPO+ HD treatment up to 4 h; thereafter, PPO+ LD showed the highest concentration up to 24 h which was comparable with the PPO- LD treatment. Auto-oxidation was more prevalent at the earlier time points for PPO- HD than PPO- LD, but the inverse relationship was shown at 24 h. The experiment highlights the temporal denaturation of PPO and the role of auto-oxidation in the formation of PBP in damaged red clover during wilting.