Some Biochemical properties of polyphenoloxidase from spearmint (Mentha arvensis)

Polyphenoloxidase (PPO; EC 1.14.18.1) extracted from Mentha arvensis leaves was isolated by (NH4)2SO4 precipitation and extensive dialysis. Its optimum pH and temperature varied with the substrate. The PPO showed activity with various diphenols. Km values were found 0.825, 0.928 and 7.41mM for caffeic acid, 4-methylcatechol and catechol, respectively. On heat-inactivation, half of the activity was lost after 60 and 15 sec at 70 and 75ºC, respectively. Measuring of residual activity showed a stabilizing effect of sucrose at various temperatures with activation energy (Ea) for inactivation increasing with sucrose concentration from 0 to 40 percent (w/w). Ea values of 78.13; 80.37; 82.79 and 81.00 kJ/Mol were found for 0, 15; 30 and 40 percent sucrose, respectively. PPO was inhibited by ascorbic, benzoic, cinnamic, ferulic, p-coumaric, protocatechuich acids, sodium metabisulfite, pyrogallol and resorcinol. The Ki values showed that ascorbic acid was the most effective inhibitor. The type inhibition was determined for each inhibitor.

Polifenoloxidase (PPO, EC 1.14.18.1) extraída de folhas de Mentha arvensis foi isolada por precipitação com (NH4)2SO4 e diálise extensiva. Seu pH e temperatura ótimos variaram com o substrato. A PPO apresentou atividade com vários difenóis. Valores de Km foram 0,825; 0,928 e 7,41 mM para ácido caféico, 4-metilcatecol e catecol, respectivamente. Na inativação térmica, 50 por cento da enzima foi inativada após 60 e 15 segundos a 70 e 75ºC, respectivamente. A medida de atividade residual mostrou um efeito estabilizante de sacarose a várias temperaturas e uma energia de ativação (Ea) para inativação aumentando com a concentração de sacarose de 0 a 40 por cento (p/p). Valores de energias de ativação de 78,13; 80,37; 82,79 and 81,00 kJ/Mol foram encontradas para 0, 15, 30 e 40 por cento de sacarose, respectivamente. A PPO foi inibida pelos ácidos ascórbico, benzóico, cinamico, ferulico, p-cumárico, protocatéquico, além de metabisulfito de sódio, resorcinol e pirogalol. Os valores de Ki mostram que o ácido ascórbico foi o mais efetivo inibidor. O tipo de inibição foi determinado para cada inibidor.

Comparison between heat inactivation and DEAF trisacryl methods for the determination of urinary N-acetyl-ß- D-glucosaminidase isozyme in normal subjects and patients with glomerulonephritis.

We report a study on NAG-B isozyme determined by heat inactivation and DEAE trisacryl methods in 231 glomerulinepgriyis patients and normal subjects There was a good correlation between the NAG-B activities measured by both methods (r = 0.902). The NAG-B activity determined by DEAE trsacryl method was significantly higher than the heat inactivation method. The mean + SD NAG-B values of 58 normal Thai subjects, 69 Patients with idiopathic nephrotic syndrome and 50 with SLE were 0.30 + 0.23, 5.12 + 8.30 and 3.25 + 3.45 U/g creatinine respectively. There was no significant difference between sex and random single and 24 hour urine. The patients had a significantly higher NAG-B value than normal controls bur there was no difference between nephrotic syndrome and SLE. From our experience, NAG isozyme isozyme activity determination by this heat inactivation method is accurate, inexpensive, rapid and easy to perform, and therefore, it is a suitable procedure for NAG isozyme measurement.