59 



methanol; pH 8.3) for 15 min. Electro-transfer was performed according to 

 the Trans-Blot Electrophoretic Transfer Cell Instruction Manual (Bio-Rad, 

 1989) at a constant voltage of 50 V for 1.5 hr using Towbin buffer as the 

 electrolytic buffer. Complete transfer of proteins was verified by 

 staining the gel and the nitrocellulose membrane with a Coomassie blue 

 solution. 



The nitrocellulose membrane following electro-transferring was 

 rinsed with phosphate-buffered saline (PBS) 3 times, and then incubated 

 with Blotto/Tween blocking solution (5% w/v nonfat dry milk, 0.2% v/v 

 Tween 20, and 0.02% w/v NaNg in PBS; Harlow and Lane, 1988) at ambient 

 temperature with agitation for 2 hr. After washing twice for 5 min each 

 in PBS, the membrane was incubated overnight in the primary antibody (IgY) 

 solution (10 Mg/mL). Following washing with 4 changes of PBS for 5 min 

 each, the membrane was treated for 1 hr with the secondary antibody 

 (antichicken IgG-alkaline phosphatase conjugate, Sigma) at a dilution of 

 1/2000. The membrane was then washed again with PBS as previously 

 described, and incubated with 100 mL alkaline phosphatase buffer (100 mM 

 NaCl, 5 mM MgCl^, and 100 mM Tris; pH 9.5) containing 0.016% 

 bromochloroindolyl phosphate (BCIP) and 0.032% nitro blue tetrazolium 

 (NBT) (Harlow and Lane, 1988) for 45 min. The reaction was stopped by 

 rinsing the membrane with PBS containing 20 mM EDTA. 



Spectropolarimetric Analysis of PPO 



The circular dichromic spectra of PPO was scanned at the far UV (250 

 - 200 nm) range using a Jasco J-20 automatic recording spectropolarimeter 

 (Japan Spectroscopic Co., Tokyo, Japan). A 1.0-cm Suprasil (Helma Cells) 



