Carpenter J F, Crowe L M, Crowe J H
Biochim Biophys Acta. 1987 Jan 20;923(1):109-15. doi: 10.1016/0304-4165(87)90133-4.
Phosphofructokinase purified from rabbit skeletal muscle is fully inactivated after freeze-drying and dissolution. The addition of trehalose or maltose to the enzyme solution prior to freeze-drying results in a recovery of up to 80% of the original activity. Slightly less stabilization is imparted by sucrose, whereas glucose and galactose at concentrations up to 500 mM are relatively ineffective at protecting phosphofructokinase. Addition of ionic zinc to enzyme-sugar mixtures prior to freeze-drying greatly enhances the stabilization imparted by the above sugars. This effect is not simply due to the summation of the individual protective capacities of zinc and the sugar. Zinc alone affords no protection, but a high degree of stabilization is achieved when zinc is added to a sugar solution, even when the sugar is at a concentration at which, by itself, it is totally ineffective. In the presence of a constant sugar concentration (100 mM), freeze-dry stabilization of phosphofructokinase is increased as the concentration of zinc is increased. When the zinc concentration is held constant (0.9 mM) and the sugar concentration varied, the maximum stabilization is noted with less than 200 mM sugar. At higher solute concentrations the degree of enhancement decreases such that with 500 mM sugar the addition of zinc results in only a slight increase in protection. Several other organic solutes (proline, 4-hydroxyproline, glycine, trimethylamine N-oxide, glycerol and myo-inositol) that afford cryoprotection to phosphofructokinase, an effect enhanced by the addition of zinc, do not stabilize the enzyme during freeze-drying, even if zinc is present. The addition of ionic copper, cadmium, nickel, cobalt, calcium and manganese to trehalose-phosphofructokinase solutions prior to freeze-drying also increases the percentage of activity recovered after dissolution. Magnesium is ineffective in this respect.
从兔骨骼肌中纯化得到的磷酸果糖激酶在冻干和溶解后会完全失活。在冻干前向酶溶液中添加海藻糖或麦芽糖，可使原始活性恢复高达80%。蔗糖提供的稳定性略低，而浓度高达500 mM的葡萄糖和半乳糖在保护磷酸果糖激酶方面相对无效。在冻干前向酶 - 糖混合物中添加离子锌可大大增强上述糖类提供的稳定性。这种效应并非简单地由于锌和糖各自保护能力的总和。单独的锌没有保护作用，但当锌添加到糖溶液中时，即使糖的浓度本身完全无效，也能实现高度的稳定性。在恒定糖浓度（100 mM）存在下，随着锌浓度的增加，磷酸果糖激酶的冻干稳定性增强。当锌浓度保持恒定（0.9 mM）且糖浓度变化时，在糖浓度低于200 mM时观察到最大稳定性。在较高溶质浓度下，增强程度降低，以至于在500 mM糖的情况下添加锌仅导致保护作用略有增加。其他几种对磷酸果糖激酶具有冷冻保护作用（添加锌可增强这种作用）的有机溶质（脯氨酸、4 - 羟脯氨酸、甘氨酸、氧化三甲胺、甘油和肌醇），即使存在锌，在冻干过程中也不能使酶稳定。在冻干前向海藻糖 - 磷酸果糖激酶溶液中添加离子铜、镉、镍、钴、钙和锰，也会增加溶解后恢复的活性百分比。镁在这方面无效。
