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Destetrapeptide insulin (DTI, human insulin with B27-30 removed) was obtained from a monomeric precursor (MIP) expressed in Saccharomyces cerevisiae through tryptic transpeptidation in the presence of synthetic tetrapeptide Gly-Phe-Phe-Tyr. The in vivo biological activity of DTI, determined by mouse convulsion assay, is 22 IU/mg. Its binding activity with insulin receptor on human placental membrane is 80% and its in vitro biological activity, determined by free fat cell assay, is 77%. Compared with native insulin, DTI molecules do not associate in solution but exist in the monomeric form, thus leading to its rapid utilization in vivo.

In view of the similarity of the charge distribution between fibrin A alpha 148-161 and A chain 149-157 of urokinase, the latter might compete with fibrin A alpha 148-161 when single chain pro-urokinase is converted to double chain urokinase. To test this, the stretch of urokinase A chain 135-157 was separated from the low molecular weight urokinase, a competitive binding between this stretch and fibrin to tPA kringle-2 was shown by radio-binding assay. The inhibition of the stretch on the fibrin stimulated activation of plasminogen was demonstrated in the caseinolytic system. The synthesized novapeptide urokinase A chain 149-157 (R-peptide) showed a significant inhibition on the activation of plasminogen in the presence of fibrin. By contrasting finely with R-peptide, a synthesized novapeptide in which Arg154 and Arg156 were replaced by Asp (D-peptide) did not show any inhibition effect on the fibrin stimulated activation of plasminogen by tPA. These results suggest that the positively charged residues in the stretch 149-157 of urokinase are crucial for the inhibition of fibrin binding with the kringle domain of urokinase.

Des-(B25-B30)-hexapeptide-insulin with B23-glycine replaced by D-alanine was prepared by a combination of enzymic and non-enzymic syntheses. The purified product was homogeneous in polyacrylamide-gel electrophoresis and could be crystallized. The biological activity in vivo of crystalline [B23-D-Ala]des-(B25-B30)-hexapeptide-insulin was determined as 58% of that of standard pig insulin (27 i.u./mg).