-, ;'^-^'-- 



60 

 cuvette with a 1.0-cm light path was used. Four-mL of PPO (10 - 20 ^g/mL) 

 in 0.5 mM sodium phosphate buffer (pH 6.5) was analyzed at ambient 

 temperature. Calculations of the secondary structures were carried out by 

 computer analysis of the spectra using the SSE program (Japan 

 Spectroscopic Co., 1985) with myoglobin, cytochrome c, ribonuclease A, 

 lysozyme, and papain as references. 



r. 



Results and Discussinn 

 SDS-PAGE Profile of PPn 



Various enzyme subunits with different molecular masses were 

 observed for crude mushroom (7 isozymes), potato (5 isozymes), lobster (3 

 isozymes), white shrimp (2 isozymes), and brown shrimp (2 isozymes) PPO 

 preparations after the nondenaturing preparative polyacryl amide gel was 

 stained with DL-DOPA. After subjecting to SDS-PAGE (without treatment of 

 ^-mercaptoethanol), mushroom and potato PPO chosen for this study were 

 shown to have a subunit of lower molecular mass than crustacean (lobster, 

 white shrimp, and brown shrimp) PPO, estimated as 66 and 148 kD, ^ 

 respectively (Figure 10). These are in agreement with the previously 

 reported data of Anisimov et al. (1978) and Bouchilloux et al . (1963). As 

 to lobster, white shrimp, and brown shrimp PPO, the molecular masses were 

 determined as 200, 190, and 190 kD, respectively. Brown shrimp PPO of 190 

 kD was close to that of the Gulf brown shrimp PPO (210 kD) (Madero and 

 Finne, 1982). However, the molecular mass of white shrimp PPO determined 

 in this study varied with that reported previously by Simpson et al . 

 (1988). The use of different preparation and analytical methods in these 

 studies could have contributed to such discrepancy. ■ 



