Amino terminal group in chymotrypsinogen.

Bovine chymotrypsinogen Q! was examined for N-terminal groups by Rovery et al. (l), using both the dinitrophenyl (DNP) and the phenyl isocyanate techniques (2, 3). Their failure to find evidence for any such group, together with the absence of a C-terminal residue reactive towards carboxypeptidase (4), suggested that this protein consists of one or more cyclic peptide chains. Evidence has now been obtained that one amino group of a cystine residue is free in chymotrypsinogen. No ether-extractable DNP-amino acid could be detected in hydrolysates of DNP-chymotrypsinogen in amounts corresponding to more than 0.1 residue per mole. However, after long periods (40 hours) of hydrolysis in 5.7 N HCl, and also after hydrolysis for 6 hours in 12 N HCl, an ether-extractable substance was found which moved in the tert-amyl alcohol chromatographic system (5) slightly faster than DNP-threonine, but was distinguished from DNP-amino acids by having a brownish rather than a yellow color. This brown substance was not found when the DNP protein was oxidized with performic acid (6) before hydrolysis. Of the DNP derivatives of amino acids likely to be affected by this treatment, bis-DNPcystine was found to give rise to the same brown material on heating in HCl, whereas DNP-methionine and DNP-tryptophan did not. MonoDNP-cystine behaved like the bis-DNP derivative in this respect, as was to be expected from the interconvertibility of the two in strong acid (7). The nature of the brown substance is not known. Its ultraviolet absorption spectrum showed a broad maximum near 400 rnp, instead of the sharp peak near 350 m,u characteristic of DNP-amino acids. It was destroyed by performic acid without giving rise to DNP-cysteic acid. Decomposition products of DNP derivatives of cystine have also been observed by Porter and Sanger (8) and by Davoll et al. (9). Yields of the brown substance were far from reproducible, and for quantitative work the N-terminal cystine was estimated as DNP-cysteic acid after oxidation of the DNP protein with performic acid.