Abstract
In order to develop a radioimmunoassay for rat C-peptide, rat insulinoma, which contains large quantities of insulin and was expected also to contain large quantities of C-peptide, was chosen as a starting material. However, the tissue was found not to contain extractable C-peptide. Instead, a novel peptide (rat insulinoma peptide, RIP) was isolated. Rat insulinoma peptide (RIP) which appears to be either a fragment of, or an altered rat C-peptide was isolated and purified by dialysis. The purity of this peptide was investigated using polyacrylamide gel electrophoresis with sodium dodecyl sulphate, isoelectric focussing, and high performance liquid chromatography. RIP may contain two peptides similar to each other but differing in their isoelectric points. The molecular weight of RIP was found to be 1,982 daltons by fast atom bombardment mass spectrometry giving a chain length of approximately 22 amino acid residues. From information obtained using radioimmunoaasay employing antiserum R901, RIP appears to share a common C-terminus with rat C-peptide. A radioimmunoassay for RIP was developed using the purified RIP as immunogen and for standards and tracers. An indirect enzyme linked immunosorbent assay (ELISA) for rat insulinoma peptide was developed using purified RIP for immunogen and semi-purified RIP as a standard. Rat C-peptide I and II were successfully synthesised using the technique of solid phase peptide synthesis. The crude synthetic peptides were purified by dialysis, and their purity was assessed by high performance liquid chromatography. The molecular weight of these synthetic peptides was determined by fast atom bombardment mass spectrometry to be 3,183 daltons. These two synthetic peptides can be detected by rat C-peptide I radioimmunoassay employing antiserum R901. A radioimmunoassay for rat C-peptide I was developed using synthetic rat C-peptide I for immunogen, standard and tracer. The rat C-peptide I and II antisera were shown to produce positive staining of the islets of Langerhans of normal rat pancreas.