The ultraviolet circular dichroism of di-isopropylphophoryl-subtilisins Carlsberg and Novo (EC 126.96.36.199) has been examined as a function of pH. The CD of these enzymes below 260 nm is invariant over the pH interval 4 to 12, below or above which spectral changes occur suggesting a transition to a random coil form. Above pH 8 contributions due to the ionization of tyrosyl residues appear in the CD above 260 nm as bands shifted to longer wavelengths. Three independently titratable components, obtained by matrix rank analysis, account for the observed CD spectral changes above 260 nm of Dip-subtilisin Carlsberg in the pH interval 8 to 12. By contrast, two components were derived for the Novo enzyme. The identities of the matrix rank component were surmised from their apparent pK1 values. One component of both subtilisin enzymes corresponds to the CD of the "buried" or irreversibly titratable tyrosyl residues of the enzyme. The other matrix rank components correspond to the CD of the "exposed' or freely ionizable tyrosyl residues. These residues are optically active only in the ionized state. Two types of "expressed" tyrosyl residues, arising because of differing sensitivity to the ionization of the "partially buried" or abnormally titrating tyrosyl residues, are evident in Dip-subtilisin Carlsberg. A pH-induced local conformational change in this enzyme is proposed to account for this behavior. The "partially buried" tyrosyl residues of both subtilisins appear to be devoid of optical activity in either the tyrosyl or tyrosylate form.
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