The Use of Affinity Chromatography in Determining the Sites of Protein‐Protein Interaction Relative to the Binding Sites of Substrates in Gramicidin S Synthetase

Columns containing one of the amino acids in gramicidin S bound through its carboxyl group to the amino group of amino‐alkyl‐Sepharose (CNBr‐activated Sepharose + 3,3′‐diaminodipropylamine) have been prepared and were found to exhibit affinity for the corresponding enzymes (light or heavy) of gramicidin S synthetase. The d‐phenylalanyl ligand will bind the light enzyme and the l‐propyl, l‐valyl, l‐ornithyl or l‐leucyl ligand, the heavy enzyme. These columns permitted a study of the interaction of the light and heavy enzyme. The results show that the area of the light enzyme which has affinity for the heavy enzyme is that part which activates phenylalanine. The area of the heavy enzyme which interacts with the light enzyme is not that part of the enzyme which activates proline. However, it is probably located close to the activation sites for proline and leucine. The columns, in particular with proline as ligand, provide a quick and convenient method for the separation of the light and heavy enzyme of gramicidin S synthetase.