Primary Study On The Biochemical Characteristics Of Tyrosinase In Housefly, Musca Domestica

Biochemical characteristics of tyrosinase in Musca domestica were studied. The activity of tyrosinase in different parts of third-day adults followed the order: head>thorax > abdomen and the activity of tyrosinase in different development stages of Musca domestica followed the order : third instar larvae>second instar larvae>first instar larvae>adult>pupa. The effect of pH and temperature on the activity of tyrosinase was also studied, using catechol as substrate and the optimum pH and temperature for reaction were found. The optimum pH was 7.4 and the temperature was 45℃, respectively. The tyrosinase of Musca domestica was partially purified by ammunium sulfate, PEG-400,PEG-10000 and PEG-20000 fractionation, respectively. Results showed that the purification by ammunium sulfate and PEG-400 was better than that by PEG-10000 and PEG-20000 , The tyrosinase was purified to 16.26-fold after G-25 and G-150 column were used to purify 15%-20% typosinase precipitated by ammunium sulfate ( dialysed ), which was 4.37-fold to the purification by ammunium sulfate. The relationship between the activity of tyrosinase kept in different temperature and time was measured. When stored at 22℃ ,4℃ and -80℃, the prepared tyrosinase could keep its best activity for 12 h,72 h and 72 h, respectively ; and while stored at - 20℃, the activity of tyrosinase began to reduce after 3 days. Phenylthiourea was the best inhibitor of tyrosinase and phoxim and parathion followed. The results of inhibition kinetics indicated that the inhibition of phenyl thiourea, phoxim and parathion to tyrosinase all belonged to mixed type, as the inhibition line of phoxim and parathion met with the line of the control in the second quadrant and the intersection of the inhibition line of phenyl thiourea the control's was in the third quadrant. The Km vakyes of tyrosinase inhibited by phoxim, parathion and phenyl thiourea were 0.9447, 0.9817 and 0.6269mM, respectively and Vmax were 0.50153, 0.49918 and 0.22910 OD/min/mg.pro, respectively. The Km values of of tyrosinase in the larvae increased, but the Vmax decreased after the enzyme was inhibited by phoxim or parathion. However, both Km and Vmax decreased after the tyrosinase was inhibited by phenyl thiourea. The activity of tyrosinase inhibited by parathion and phenyl thiourea decreased with the time, while it showed no obvious difference when inhibited by phoxim and ethonal. The K, values of tyrosinase to phoxim, parathion, phenyl thiourea and ethanol ere 110.6 M-1.min-1,434.8M-1.min-1, 4.4×105 M-1.min-1 and 216.8 M-1.min-1, respectively, which showed high affinty of phenyl thiourea, parathion, ethanol and phocim to tyrosinase.