102 

 The inhibitory effect of kojic acid on the enzyme activity in oxidizing 

 DL-DOPA in these systems was determined by adding 50 /xL of kojic acid at 

 0.56 or 1.12 mM to the cuvette containing PPO and sodium phosphate buffer 

 (pH 6.5). The mixture was pre-incubated at 37°C for 15 min. Following the 

 equilibration to ambient temperature, DL-DOPA was added and the reaction 

 was monitored at 475 nm (25°C) for 10 min. All the assays to determine 

 enzyme activity, the inhibitory effect of kojic acid on the various 

 enzymes, and the enzyme kinetics were conducted at least three times with 

 three different sample preparations. 



Effect of Pre- incubation Temperature on PPO Inhibition bv Ko.iic Acid 



A reaction mixture containing 950 ill of 0.13 mM kojic acid in 0.05 

 M sodium phosphate buffer (pH 6.5) and 50 nl mushroom tyrosinase (0.5 

 mg/mL) or spiny lobster PPO (0.5 mg/mL) was pre-incubated for 15 min in a 

 cuvette at 0°, 25°, or 37°C. After the mixture was equilibrated to ambient 

 temperature, 500 /xL 10 mM DL-DOPA in 0.05 M sodium phosphate buffer (pH 

 6.5) was added and the reaction monitored at 475 nm (25°C) for 5 min. For 

 controls, kojic acid was replaced by phosphate buffer. Percent inhibition 

 (I) was expressed as [(A - A*)/A] x 100, where A and A* were enzyme 

 activities in the absence and presence of the inhibitor (kojic acid), 

 respectively (Saruno et al., 1979). 



Similarly, 50 /iL potato PPO was added to a cuvette containing 500 nl 

 1 mM sodium phosphate buffer (pH 7.0) and 450 /xL 0.56 mM kojic acid. 

 After incubation at 0°, 25°, or 37°C for 15 min, and equilibration back to 

 ambient temperature, 500 /iL 20 mM catechol in 1 mM phosphate buffer (pH 

 7.0) was added. The reaction was monitored at 395 nm (25°C) for 5 min. 



