Townsend M W, DeLuca P P
Lilly Research Laboratories, Eli Lilly Company, Indianapolis, IN 46285.
J Pharm Sci. 1991 Jan;80(1):63-6. doi: 10.1002/jps.2600800116.
Ribonuclease A (RNase) has been shown to lose enzymatic activity and to form aggregates when stored in the freeze-dried form at elevated temperature. Polyacrylamide gel electrophoresis showed that the freeze-dried RNase that had lost its enzymatic activity during storage had formed aggregates that were not dissociable using both an anionic detergent and a reducing agent. Isoelectric focusing patterns of freeze-dried RNase before and after storage were quite different. The RNase that had been stored and had aggregated had become more of an acidic protein, while the RNase that was assayed immediately after freeze drying had the same pattern as non-freeze-dried RNase. This evidence indicated that the aggregates were covalently bonded together as a result of some chemical process that occurred during storage of the freeze-dried cake. Amino acid analysis of aggregates formed from RNase freeze dried in pH 10.0 phosphate buffer solutions indicated that the amino acid lysine was instrumental in the formation of the covalent bonds. Asparagine/aspartic acid and glutamine/glutamic acid may have also participated in the bonding of the aggregates.
核糖核酸酶A（RNase）已被证明，当以冻干形式在高温下储存时，会失去酶活性并形成聚集体。聚丙烯酰胺凝胶电泳显示，储存期间失去酶活性的冻干RNase形成了聚集体，使用阴离子洗涤剂和还原剂都无法使其解离。储存前后冻干RNase的等电聚焦图谱有很大不同。储存并聚集的RNase变成了更酸性的蛋白质，而冻干后立即检测的RNase具有与未冻干RNase相同的图谱。这一证据表明，聚集体是由于在冻干饼储存期间发生的某种化学过程而共价结合在一起的。对在pH 10.0磷酸盐缓冲溶液中冻干的RNase形成的聚集体进行氨基酸分析表明，氨基酸赖氨酸在共价键的形成中起作用。天冬酰胺/天冬氨酸和谷氨酰胺/谷氨酸可能也参与了聚集体的结合。
