During cold acclimation, winter rye (Secale cereale) plants accumulate pathogenesis-related proteins that are also antifreeze proteins (AFPs) because they adsorb onto ice and inhibit its growth. Although they promote winter survival in planta, these dual-function AFPs proteins lose activity when stored at subzero temperatures in vitro, so we examined their stability in solutions containing CaCl2, MgCl2, or NaCl. Antifreeze activity was unaffected by salts before freezing, but decreased after freezing and thawing in CaCl2 and was recovered by adding a chelator. Ca2+ enhanced chitinase activity 3- to 5-fold in unfrozen samples, although hydrolytic activity also decreased after freezing and thawing in CaCl2. Native PAGE, circular dichroism, and Trp fluorescence experiments showed that the AFPs partially unfold after freezing and thawing, but they fold more compactly or aggregate in CaCl2. Ruthenium red, which binds to Ca2+-binding sites, readily stained AFPs in the absence of Ca2+, but less stain was visible after freezing and thawing AFPs in CaCl2. We conclude that the structure of AFPs changes during freezing and thawing, creating new Ca2+-binding sites. Once Ca2+ binds to those sites, antifreeze activity, chitinase activity and ruthenium red binding are all inhibited. Because free Ca2+ concentrations are typically low in the apoplast, antifreeze activity is probably stable to freezing and thawing in planta. Ca2+ may regulate chitinase activity if concentrations are increased locally by release from pectin or interaction with Ca2+-binding proteins. Furthermore, antifreeze activity can be easily maintained in vitro by including a chelator during frozen storage.

The influence of the shear rate, the membrane pore size, and the age of the culture at time of harvest on transmembrane pressure (TMP) increase and membrane fouling during the microfiltration of a Chinese Hamster Ovary (CHO) cell culture supernatant was investigated. A hollow fiber microfiltration system operated at constant permeate flux was used. The highest TMP increase with filtration time was observed for the small membrane pore size (0.20 microm) operated at the higher shear rate (8,000 s(-1)). Furthermore, the high overall fouling observed with the small membrane pore size was also associated with the highest irreversible fouling and the most significant decrease of predicted open pore area. The predicted reduction in open pore area obtained with the combined pore blockage and cake formation mechanism could explain the observed TMP profiles. Based on the overall membrane fouling, the long-term irreversible fouling and the initial fouling rate, derived from an empirical curve fitting, no effect of the time of harvest was observed but a dependence of the initial fouling rate on the shear rate was identified. Treatment of the fouled membrane with water showed the presence of a more significant reversible fouling at high shear rates and increased irreversible fouling with smaller membrane pore size. It is recommended to use the large membrane pore size (0.45 microm) and the low shear rate (4,000 s(-1)) to minimize fouling associated with the soluble components of serum-free CHO cell culture supernatant.