Nature of aggregates formed during storage of freeze-dried ribonuclease A

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.