Nealon D A, Henderson A R
Clin Chem. 1977 May;23(5):816-29.
We assessed the stability--at 20, 4 --20, and --80 degrees C--of 2-mercaptoethanol, dithioerythritol, and dithiothreitol, and of semi-purified creatine kinase isoenzymes of human origin in the presence and absence of the three compounds. The isoenzymes plus the sulfhydryl compounds were also assessed an aqueous buffer, in heat-inactivated pooled sera, or in fresh pooled sera with low endogenous total creatine kinase. We also examined the influence of thawing conditions from--20 or--80 degrees C, of fast freezing, and of exposure to fluorescent light. At 20 and 4 degrees C some--SH groups are oxidized. At--20 and--80 degrees C this loss is diminished. It is a function of both temperature and diluent. For stability, creative kinase isoenzymes stored at any temperature require the presence of a suitable sulfhydryl compound. Periodic addition of fresh sulfhydryl solutions stabilized creatine kinase isoenzymes suspended in the two protein-based diluents by 10-30%; for those in an aqueous buffer such additions decreased the activity to stored creatine kinase isoenzymes. Fast freezing does not increase recovery of creatine kinase isoenzyme activity over slow freezing at--80 or--20 degrees C. Thawing should be done as infrequently as possible and should be done rapidly, at 37 degrees C, for the residual enzyme activity to be maximal.
我们评估了2-巯基乙醇、二硫赤藓糖醇和二硫苏糖醇在20℃、4℃～20℃以及-80℃下的稳定性，以及在有或无这三种化合物存在的情况下人源半纯化肌酸激酶同工酶的稳定性。在水性缓冲液、热灭活混合血清或内源性总肌酸激酶含量低的新鲜混合血清中，对同工酶加巯基化合物也进行了评估。我们还研究了从-20℃或-80℃解冻的条件、快速冷冻以及暴露于荧光下的影响。在20℃和4℃时，一些巯基会被氧化。在-20℃和-80℃时，这种损失会减少。这是温度和稀释剂共同作用的结果。为保持稳定性，储存在任何温度下的肌酸激酶同工酶都需要有合适的巯基化合物存在。定期添加新鲜的巯基溶液可使悬浮在两种基于蛋白质的稀释剂中的肌酸激酶同工酶稳定性提高10%～30%；对于在水性缓冲液中的同工酶，这种添加会降低储存的肌酸激酶同工酶的活性。在-80℃或-20℃下，快速冷冻不会比缓慢冷冻提高肌酸激酶同工酶活性的回收率。解冻应尽可能少进行，并且应在37℃快速进行，以使残留酶活性达到最大。
