153 

 value of the sample with those of the protein standards (Broad pi Kit, pH 

 3-10, Pharmacia) containing amyloglucosidase, pi 3.50; soybean trypsin 

 inhibitor, pi 4.55; j3-lactog1obulin, pi 5.20; bovine carbonic anhydrase, 

 pi 5.85; human carbonic anhydrase B, pi 6.55; horse myoglobin-acidic band, 

 pi 6.85; -basic band, pi 7.35; lentil lectin-acidic band, pi 8.15; -middle 

 band, pi 8.45; -basic band, pi 8.65; and trypsinogen, pi 9.30. 



SpectroDolarimetric Analysis of PPG 



Circular dichroic (CD) spectra of high pressure CO^-treated and non- 

 CO^-treated PPG were determined at the far UV range (250 - 200 nm) using 

 a Jasco J-20 automatic recording spectropolarimeter (Japan Spectroscopic 

 Co., Tokyo, Japan), using a 1.0-cm Suprasil (Helma Cells) cuvette with 

 1.0-cm light path. Four-mL PPG (10 Mg/mL) in 0.05 M sodium phosphate 

 buffer (pH 6.5) was used as sample and the measurement of CD spectra was 

 carried out at ambient temperature. Secondary structure calculations were 

 performed by computer analysis of the CD spectra using the SSE program 

 (Japan Spectroscopic Co., 1985) with myoglobin, cytochrome c, ribonuclease 

 A, lysozyme, and papain as CD references. 



Study of Restoration of CG . -treated PPQ Activity 



To examine the reactivation ability of PPG following CO2 (1 or 58 

 atm) treatment, a portion of CO^-treated sample was stored at -20°C in a 

 microcentrifuge tube (1.5 mL) for 6 weeks. After thawing at ambient 

 temperature, the pH was then measured using a digital pH meter. Enzyme 

 activity was determined as previously described and the assays were 

 performed weekly. Percentage relative activity was determined as (ER^./ERg) 



