Carpenter J F, Hand S C, Crowe L M, Crowe J H
Arch Biochem Biophys. 1986 Nov 1;250(2):505-12. doi: 10.1016/0003-9861(86)90755-1.
Phosphofructokinase (PFK) purified from rabbit skeletal muscle is fully inactivated after being frozen in liquid nitrogen for 30 s and thawed. The addition of 500 mM trehalose, sucrose, or proline to the enzyme solution prior to freezing results in a recovery of over 70% of the original activity after thawing. Slightly less stabilization is imparted by maltose and 4-hydroxyproline whereas glucose, glycine, inositol, and glycerol at concentrations up to 500 mM are relatively ineffective at protecting PFK. With 50 mM trimethylamine-N-oxide, almost 50% of the prefreeze activity is recovered, and this same level of cryoprotection is noted at concentrations up to 500 mM. The addition of ionic zinc to enzyme-organic solute mixtures prior to freezing greatly enhances the cryoprotection imparted by all of the solutes tested. This effect is not simply due to the summation of the individual cryoprotective capacities of zinc and the organic solute because in many instances a great degree of cryoprotection is noted when each component is present at a concentration at which, by itself, it is totally ineffective. In the presence of a constant 50 mM organic solute concentration, freeze-thaw stabilization of PFK is increased as the concentration of zinc is increased. When the zinc concentration is held constant (0.6 mM) and organic solute concentration varied, the maximum cryoprotection, in most cases, is noted with less than 50 mM organic solute. At higher solute concentrations the degree of enhancement decreases such that with 500 mM organic solute the addition of zinc results in only a slight increase in protection. The addition of ionic copper, cadmium, nickel, and cobalt to trehalose-PFK solutions prior to freezing also increases the percentage of activity recovered after thawing. Magnesium, manganese, and calcium are ineffective in this respect.
从兔骨骼肌中纯化得到的磷酸果糖激酶（PFK）在液氮中冷冻30秒并解冻后会完全失活。在冷冻前向酶溶液中添加500 mM海藻糖、蔗糖或脯氨酸，解冻后可恢复超过70%的原始活性。麦芽糖和4-羟基脯氨酸的稳定作用稍弱，而浓度高达500 mM的葡萄糖、甘氨酸、肌醇和甘油在保护PFK方面相对无效。添加50 mM三甲胺-N-氧化物后，解冻后可恢复近50%的冷冻前活性，在浓度高达500 mM时也能观察到相同水平的冷冻保护作用。在冷冻前向酶-有机溶质混合物中添加离子锌可大大增强所有测试溶质的冷冻保护作用。这种效应并非仅仅是锌和有机溶质各自冷冻保护能力的简单相加，因为在许多情况下，当每种成分的浓度单独存在时完全无效，但同时存在时却能观察到很强的冷冻保护作用。在有机溶质浓度恒定为50 mM的情况下，随着锌浓度的增加，PFK的冻融稳定性增强。当锌浓度保持恒定（0.6 mM）而有机溶质浓度变化时，在大多数情况下，有机溶质浓度低于50 mM时可观察到最大的冷冻保护作用。在较高溶质浓度下，增强程度会降低，以至于在有机溶质浓度为500 mM时添加锌只会使保护作用略有增加。在冷冻前向海藻糖-PFK溶液中添加离子铜、镉、镍和钴也会增加解冻后恢复的活性百分比。镁、锰和钙在这方面无效。
