FRACTIONATION AND PROPERTIES OF AMINOPEPTIDASE B DURING PURIFICATION AND STORAGE

Purified preparations of rat liver aminopeptidase B were found to display heterogeneity which was demonstrated in isoelectric focusing and in electrophoresis on polyacrylamide columns. Most of the heterogeneity was generated during the purification of the enzyme. Acid treatment at pH 5.1 and ammonium sulphate fractionations caused considerable heterogeneity and the presence of thiols reduced it. Ampholine ampholytes themselves did not cause the generation of the heterogeneity but isoelectric focusing rendered its demonstration possible. A special type of heterogeneity was observed when aged preparations of the enzyme were found to undergo aggregation. The aggregate forms moved slowly or not at all in electrophoresis on polyacrylamide. The pIs of the main forms appearing in isoelectric focusing were around 5 and 7. The aggregate forms had a pI below 4. The enzymic properties of the different forms, whether appearing in isoelectric focusing, during ageing, or in purification were essentially similar, indicating that the heterogeneity involved changes only in those amino acid residues which were not responsible for substrate binding or catalysis.