The involvement of tyrosyl and amino groups in the interaction of trypsin and a soybean trypsin inhibitor.

Modtication of the tyrosyl and amino groups of trypsin and soybean trypsin inhibitor (Kunitz) with N-acetylimidazole before and after their combination was used to assess the role of these amino acids in the binding process. Trypsin was inactivated by au excess of the inhibitor and the complex isolated by chromatography on Sephadex G-75, at pH 6.8. Prior to complex formation, trypsin and the inhibitor each possessed 4 reactive tyrosyl residues, but after complex formation only 4 of the expected 8 reacted with N-acetylimidazole. Trypsin and the inhibitor had four and three acetylatable amino groups, respectively, but only two of these were reactive in the complex. These results indicated that four tyrosyl and five amino groups had been prevented from reacting with N-acetylimidazole because of their involvement in the formation of the complex. Modified but fully active trypsin and inhibitor could be recovered from the acetylated complex by chromatography on sulfoethyl Sephadex at pH 2.6. Subsequent analysis of each of these isolated components revealed that 2 of the 4 shielded tyrosyl residues of the complex had been derived from trypsin and 2 from the inhibitor. Of the five shielded amino groups in the complex, three were contributed by trypsin and two by the inhibitor.