Susanne Linde

Biological potency and binding affinity of monoiodoinsulin with iodine in tyrosine A14 or tyrosine A19.

Summary Monoiodoinsulin with 99% of the iodine in the A chain was separated in two bands by disc-electrophoresis using long polyacrylamide gel rods. The bands contained monoiodoinsulin with the iodine in tyrosine A14 and tyrosine A19, respectively. The biological potency and binding affinity of A14 [ 127 I]monoiodoinsulin on rat adipocytes was indistinguishable from that of insulin, whereas the A19 derivative was only half as potent. Similarly, the maximum binding of “tracer” A19 [ 125 I]monoiodoinsulin to adipocyte receptors was only half of that obtained with A14 [ 125 I]monoiodoinsulin.

Stable lodinated polypeptide hormones prepared by polyacrylamide gel electrophoresis

Iodination of several insulin and proinsulin preparations, human growth hormone and bovine pancreatic polypeptide was performed using H2O2 and lactoperoxidase or chloramine T. The iodination mixtures were fractionated by polyacrylamide gel electrophoresis at pH 9.15 in long gel rods followed by simple elution of the iodinated products from thin gel slices. With this method 125I tracers with long shelf life and high specific activity suitable for radioimmunoassays could easily be obtained.

[24] Preparation of stable radioiodinated polypeptide hormones and proteins using polyacrylamide gel electrophoresis

Publisher Summary This chapter discusses the preparation of stable radioiodinated polypeptide hormones and proteins using polyacrylamide gel electrophoresis. Radioactively labeled polypeptide hormones and proteins are widely used as tracers in radioimmunoassays and receptor studies. The peptide or protein is most easily labeled using iodination with 125 I or 131 I. The radioactive iodine is substituted in the tyrosine groups of the peptide as monoiodotyrosine or di-iodotyrosine, resulting in a heterogeneous mixture of iodine-substituted molecules plus native peptide and unreacted iodide. The iodination mixture has to be fractionated to obtain a well-characterized iodinated product to be used as a tracer. An ideal tracer should retain the full biological activity or immunological reactivity of the native peptide hormone or protein and have a high specific activity and a long shelf life. Studies in the laboratory have shown that it is possible to fractionate iodinated insulin preparations into homogeneous monoiodoinsulin derivatives by polyacrylamide gel electrophoresis using long rods. The same fractionation technique has been useful for the preparation of a number of radioiodinated polypeptide hormones and proteins.

Separation and quantitation of serum proinsulin and proinsulin intermediates in humans

Two reversed-phase high-performance liquid chromatographic (RP-HPLC) systems were developed for the separation of human insulin, proinsulin and the major proinsulin intermediates. The individual components were quantified using two enzyme-linked immunosorbent assays for insulin and proinsulin immunore-active material (PIM) after (passive) evaporation of the organic modifier. Serum samples from normal subjects and patients with non-insulin-dependent diabetes mellitus were immunopurified and analysed in one of the RP-HPLC systems. The proportion of PIM relative to insulin immunoreactive material was higher in the diabetic patient compared with that in the normal subject. In both, PIM was heterogeneous, consisting of intact proinsulin and des-proinsulin intermediates.

Reversed-phase high-performance liquid chromatographic analyses of insulin biosynthesis in isolated rat and mouse islets

Two RP-HPLC systems were developed for the separation of the products of the conversion of proinsulin into insulin in rat and mouse islets, including proinsulin I and II. Peaks were identified by microsequencing and radiosequencing. It was confirmed that mouse C-peptide I has a two amino acid deletion compared to rat C-peptide I. A marked species difference in the ratio between insulin I and II was observed, i.e., 2:1 in the rat and 1:2 in the mouse. Pulse-chase experiments in rat islets have demonstrated that the ratio between insulin I and II in newly synthesized insulin is higher than that of the stored insulin, indicating a slower conversion rate of proinsulin II compared to proinsulin I.

Separation, isolation and characterization of the four monoiodinated insulin tracers using reversed-phase high-performance liquid chromatography

Baseline separation between insulin and insulin monoiodinated in Tyr A14, A19, B16 and B26 can be obtained using isocratic elution from a C18 column with triethylammonium trifluoroacetate-acetonitrile and the iodinated insulin derivatives can be isolated by lyophilization. Compared with similar tracers purified and isolated by disc electrophoresis/ion-exchange chromatography, the reversed-phase high-performance liquid chromatographically purified tracers are more homogeneous but show reduced binding affinity to adipocytes.

Reversed-phase high-performance liquid chromatography of insulin and insulin derivatives : A comparative study☆

Abstract The reversed-phase separation of crystalline insuline (I) and monoiodoinsulins (II) has been investigated, with respect to the effects of buffer, substitution group, pore-size and column support backbone. The separations were performed either isocratically (for II) or by gradient elution with very narrow gradients (for I). Fourteen reversed-phase columns, the majority being silica-based, were investigated, and three main result emerged. (1) Trifluoroacetic acid is unsuitable as a buffer for this type of analysis, whereas trialkylammonium phosphates are very suitable. (2) The separation between the major components in crystalline insulin was comparable in the main for all the columns tested except one. However, the ability to distinguish between the numerous minor components (co-extracted with insulin peptide) varied a great deal between the columns. (3) In an optimized buffer system only three columns were able to separate insulin peptide and the four monoiodoinsulin isomers; all three were 80—100-A silica-based C 18 columns.

Reversed-phase high-performance liquid chromatographic separation of the four monoiodoinsulins: effect of column supports, buffers and organic modifiers

Abstract The separation of mono- and diiodoinsulins has been performed using various C18 columns (LiChrosorb and Vydac), organic modifiers (acetonitrile, 2-propanol and ethanol) and trialkylammonium buffers at various pH values. One system (LiChosorb—2-propanol—triethylammonium formate, pH 6.0) allows complete separation between unlabelled insulin, monoiodoinsulins and diiodoinsulins. The more- or-less reduced binding affinity of the reversed-phase high-performance liquid chromatographic purified tracers is most likely caused by column bleeding.

Binding affinity of monoiodinated insulin tracers isolated after reversed-phase high-performance liquid chromatography

Insulin and insulin monoiodinated in tyrosine A14, A19, B16 and B26 can be separated using reversed-phase high-performance liquid chromatography on a number of C18 columns eluted with acetonitrile containing triethylammonium phosphate or acetate buffers. The monoiodoinsulins can be isolated using lyophilization, gel chromatography, or Sep-Pak purification. Compared with similar tracers purified and isolated by disc electrophoresis-ion-exchange chromatography, the resulting binding affinities to adipocytes of the purified tracers are more or less reduced dependent on the choice of column support, buffer, separation temperature, and isolation procedure.

Use of polymeric reversed-phase columns for the characterization of polypeptides extracted from human pancreata

The high-performance liquid chromatographic (HPLC) behaviour of two different styrene-divinyl-benzene-based reversed-phase (RP) columns was evaluated using crude acetic acid extracts from normal and diabetic human pancreata as samples. Acetic acid gradients in water and acetonitrile gradients in triethylammonium phosphate (TEAP) and trifluoroacetic acid (TFA) were used as mobile phases, and comparisons were made with a silica-based C4 column. When two different polymeric RP columns were eluted with acetic acid gradients in water, surprisingly similar HPLC profiles of the pancreatic extracts were obtained. Elution of the polymer-based columns with acetonitrile gradients in TFA or TEAP resulted in changes in the polypeptide selectivity of these columns, in parallel with that of a silica-based C4 column eluted under similar conditions, indicating the general usability of polymeric columns for RP-HPLC of peptides and proteins. The pronounced difference in composition between normal and diabetic samples, which also was demonstrated after size-exclusion chromatography (SEC) on a silica-based and an agarose-based high-performance SEC column, was found to be related to the different ischaemia times for the two types of pancreata.