Isolation of some crystalline yellow peptides from enzymic digests of dinitrophenyl insulin and dinitrophenyl trypsinogen.

Previous work has shown that the peptide-like growth factor strepogenin probably occurs in certain proteins such as insulin and trypsinogen at the amino end of the protein (l), and that it is liberated by suitable enzymic digestion. This conclusion suggested a means for attacking the almost insurmountable task involved in separating the one compound strepogenin from the mixture of numerous peptides and other cleavage products which result from the enzymic digestion of a protein. If the protein were converted to the dinitrophenyl (DNP) derivative by reaction of its amino groups with 2,4-dinitrofluorobenzene (2), the strepogenin of which the amino group is exposed should be converted into DNP strepogenin, which might then be liberated during enzymic digestion. A rclatively small number of yellow, DNP compounds should result from such a digestion, because the number of amino groups of the proteins is small. Furthermore, the DNP derivatives are no longer amphoteric substances, but are organic acids which can be extracted into organic solvents and thus separated from the more numerous products of protein cleavage. This latter property very materially increases the scope of means available for successful separation of mixtures. For these reasons the isolation of pure, crystalline yellow cleavage products of DNP insulin and DNP trypsinogen has been attempted. Even though the biological inactivity of DNP strepogenin rendered impossible the direct determination of which, if any, of the isolated materials might be the derivative of the growth factor, the value of the study to the problem of the exact chemical structure of proteins seemed sufficient reason for proceeding. Since much evidence points to the conclusion that strepogenin is a derivative of glutamic acid (3), the presence of this amino acid in one of the cleavage products of DNP insulin might indicate a relationship to strepogenin. However, all yellow peptides isolated contained this amino acid, and so no deduction could be made except that glutamic acid is very near the amino end of some of the pcptide chains in this protein.