»*t 



105 

 dopaquinone was also studied by adding 0.6 mL 5.63 mM kojic acid in 0.05 

 M sodium phosphate buffer (pH 6.5) to a mixture containing 0.2 mL mushroom 

 PPG (0.5 mg/mL) and 1.2 mL 10 mM DL-DOPA. Following incubation at ambient 

 temperature for 30 min, the solution was scanned as previously described. 

 An similar study was also conducted on spiny lobster PPO. 



The effect of kojic acid on dopaquinone formation was further 

 investigated using thin-layer chromatography. One-mL mushroom PPO (0.5 

 mg/mL) was mixed with 1.0 mL 5 mM DL-DOPA in 0.05 M sodium phosphate 

 buffer (pH 6.5) and the reaction was allowed to proceed at ambient 

 temperature for 2 hr. Following the reaction, 0.5 mL kojic acid (1 mg/mL) 

 in 0.05 M sodium phosphate buffer (pH 6.5) was added to 0.5 mL aliquot of 

 the reaction mixture and incubated at ambient temperature. After 2 hr, 

 80-/iL aliquots were spotted on TLC plates (20 x 20 cm Redi/plt Sil, Gel G, 

 Fisher) and the plates developed using a butanol -acetic acid-water (4: 1: 

 5, v/v) solvent system. An equivalent volume of 10 mM DL-DOPA in 0.05 M 

 sodium phosphate buffer (pH 6.5) was used as standard. The 

 chromatographic pattern was examined and the R^ value for each compound was 

 determined after spraying the plate with a ninhydrin reagent (Sigma). For 

 control sample, an equivalent volume of phosphate buffer was in place of 

 kojic acid. 



Enzymatic Activities of Ko.iic Acid-treated PPO ^ 



Two-mL of mushroom, white shrimp, and lobster PPO was individually 

 incubated at ambient temperature for 30 min with 0.5 mL kojic acid (0.56 

 or 1.12 mM) in 0.05 M sodium phosphate buffer (pH 6.5) in a Spectra/Por 

 Membrane (Spectrum Medical Industries Inc., Los Angels, CA) with molecular 



