States of Tyrosyl Residues and Circular Dichroism of Kunitz Trypsin Inhibitor<xref ref-type="fn" rid="fn1"><sup>*</sup></xref>

Abstract

In order to clarify the origin of the side chain Cotton effects of Kunitz trypsin inhibitor, effects of acetylation of the tyrosyl residues with N-acetylimidazole, oxidation of the tryptophyl residues with N-bromosuccinimide (NBS) and of pH on the circular dichroism (CD) were studied. Spectrophotometric titration of the tyrosyl groups and the change in the inhibitor activity accompanying acetylation were also examined. Spectrophotometric titration and acetylation with N-acetylimidazole demonstrate that one of the four tyrosyl residues of Kunitz trypsin inhibitor is free on the surface of the molecule and the remaining three are buried in the interior. Acetylation of all the tyrosyl residues which is achieved in 8 or 10 M urea at 50°G prevents the refolding of the inhibitor molecule on removal of urea. The CD spectrum of native Kunitz trypsin inhibitor has negative maxima at 294, 287, 280, 271, 263, and 245 mμ and a positive maximum at 226 mμ, in addition to a negative maximum at 203 mμ which arises from the peptide conformation. The negative band at around 280 mμ decreased proportionally with the increase in the degree of acetylation. A slight change in the CD band at 226 mμ was observed with acetylation of the one free tyrosyl residue. Acetylation in 8 M urea at 50°C, followed by removal of urea, modified the buried tyrosyl residues and abolished the band at 226 mμ, indicating a large contribution of the protein conformation to this band. The negative maximum at 294 mμ is originated from the tryptophyl residues. A new peak due to ionized tyrosyl residues appeared at 247 mμ with increasing pH. From the pH dependence of the CD spectrum, it was found that Kunitz trypsin inhibitor undergoes a confonnational change below pH7 and above pH 11.5. It is noteworthy that oxidation of tryptophyl residues with NBS generates new bands due to the oxidized product.