ABSTRACT
Polyphenol oxidase which is responsible for oxidative conversion of phenolic compounds to polymers, has continued to attract the attention of scientists. Here, we report the extraction, purification and biochemical properties of polyphenol oxidase (PPO) from bitter leaf (Vernonia amygdalina). The enzyme was purified and concentrated using a non-conventional approach, aqueous two-phase partitioning (ATPS) and the biochemical properties of the purified enzyme were investigated. Substrate specificity studies revealed that the enzyme predominantly exhibits diphenolase activity. The order of substrate preference was catechol > L-DOPA > caffeic acid > L-tyrosine > resorcinol>2-naphthol > phenol. The optimum pH and temperature obtained for the enzyme using catechol as substrate were 5.5 and 50 °C respectively. The estimated Michaelis constant (Km) and maximum velocity (Vmax) for the purified vaPPO using catechol as substrate were 183 ± 5.0 mM and 2000 ± 15 units/mg protein respectively. The catalytic efficiency (Vmax/Km) of the purified vaPPO was 10.9 ± 0.03 min/mg. Na+, K+ and Ba2+ remarkably activated the enzyme and the level of activation was proportional to the concentration. The vaPPO presented stability in the presence of up to 50 mM of the different metal ions tested. In contrast, Cu2+ and NH4+ inhibited the enzyme even 10 mM concentrations. The enzyme was stable in chloroform retaining up to 60% relative activity at 50% (v/v) concentration. There was an increase in the activity (143%) of the enzyme at 30% (v/v) chloroform., revealing that vaPPO could catalyze the substrate more efficiently in 30% (v/v) chloroform. Total loss of enzyme activity was observed at 20% (v/v) concentrations of acetone, ethanol and methanol. In conclusion, the properties of the vaPPO such as its catalysis in the presence of organic solvents, metals and high temperature would be of interest in many biotechnological applications.
ABSTRACT
The study was carried out to determine the influence of proteinuria on plasma and urine concentrations of electrolytes and hematological indices in Wistar rats of different age groups. Eighty Wistar rats of both sexes were used for this study. Groups 1 and 2 each consisted of 8 one month old male and female rats; 3 and 4 had 8 three month old rats; 5 and 6 had 8 six month old rats; 7 and 8 had 8 nine month old rats; 9 and 10 had 8 twelve month old rats. The plasma sodium, potassium and calcium concentrations of 3 month old rats were significantly lower when compared with 1, 6, 9 and 12 months of age. Similarly, rats aged 3 months had significantly lower urine concentrations of sodium, potassium and calcium than rats of other age groups. A strong correlation was observed between the urine protein and urine sodium of the female rats at ages 3, 9 and 12 months but it was only significant at age 12 months (pâ¯=â¯0.105 and pâ¯=â¯0.021, respectively). Also, the female rats aged 3 and 12 months had a strong correlation between their urine protein and urine calcium (pâ¯=â¯0.002 and pâ¯=â¯0.131, respectively). The red blood cells, lymphocyte and monocyte counts of the rats increased gradually and peaked at age 9 months with a subsequent decline at 12 months of age. It was concluded that the influence of proteinuria on electrolytes was least observed in the rats aged 3 months, since they had reduced and consistent plasma and urine concentrations of electrolytes measured when compared with other age groups. This implies that long-term renal studies involving the use of rats must be carefully interpreted because of the changes in plasma and urine concentrations of electrolytes as the rats age.
