A High Performance Liquid Chromatographic Assessment of the Isolation of Bovine Proinsulin and a Synthetic Proinsulin Fragment

Abstract

High performance liquid chromatographic techniques have been used for the analysis and purification of the bovine proinsulin C-peptide fragment 34–45 (H-Val-Glu-Gly-Pro-Gln-Val-Gly-Ala-Leu-Glu-Leu-Ala-OH) (I) prepared solid phase synthetic methods. Conventional open column chromatographic methods failed to resolve the desired dodecapeptide (I)' from the des-Pro⁹-undecapeptide (II), which constituted the major solid phase synthetic deletion product. On 5- or 10- μm microparticulate reversed phase columns, these peptides are readily resolved preparatively in less than twenty minutes with simple elution systems. The elution order is in accord with that expected on the basis of hydrophobic fragmental constant summation. These HPLC techniques have been extended to permit the analytical assessment of the isolation of bovine pro-insulin and the proinsulin intermediates. The conversion of bovine proinsulin initially to the intermediates and finally to the desalanyl-insulin and the C-peptide on tryptic digestion can be followed by HPLC techniques which thus supplement alternative polyacrylamide disc electrophoretic methods.