Benny S. Welinder

Quantifying biosynthetic human growth hormone in Escherichia coli with capillary electrophoresis under hydrophobic conditions

A method has been developed which is able to quantitate the content of precursor biosynthetic human growth hormone (Pre-bhGH) in the cytosol of E. coli cells containing the gene for human growth hormone (hGH). The method uses hydrophobic C18 coated capillaries with native biosynthetic human growth hormone (bhGH) as an internal standard. This allows for highly robust and precise determinations as well as the evaluation of the presence of deamidated forms in the cytosol samples. Furthermore, by modifying the running buffer with zwitterionic surfactants and an organic modifier, it is possible to detect a related form with a three sulfur atom Cys-Cys bridge (trisulfide Pre-bhGH). Thus, a strong tool for monitoring the effect of fermentation conditions on the biosynthesis of bhGH is obtained.

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.

Recovery of polypeptides after reversed-phase high-performance liquid chromatography☆

Mass recovery of individual polypeptides may be estimated under various practical conditions. With the purpose of obtaining rapid and reliable standard procedures for recovery measurements, we have compared five individual methods utilizing a silica-based stationary phase [Nucleosil C18 (7 microns)/ammonium sulphate-perchlorate-acetonitrile, pH 3.0] and a resin-based stationary phase (TSK Phenyl 5 PW RP/ammonium phosphate-acetonitrile, pH 7.0). The recoveries of insulin (6 kilodaltons), human growth hormone (22 kilodaltons) and human serum albumin (68 kilodaltons) estimated under five different experimental conditions were found to be concordant. Variations in column load, flow-rate, gradient shape and column dwell time and addition of cyclame did not increase the (reduced) recovery of serum albumin and growth hormone.

Alternative mobile phases for the reversed-phase high-performance liquid chromatography of peptides and proteins.

The use of a high content of acetic acid as mobile phase additive for the reversed-phase high-performance liquid chromatography (RP-HPLC) of several proteins and extracts of biological tissues was evaluated for a divinylbenzene (DVB)-based stationary phase, and the separations obtained with acetic acid gradients in acetonitrile, isopropanol or water were compared with classical polypeptide RP-HPLC on silica C4 with trifluoroacetic acid (TFA)-acetonitrile. The separation patterns for recombinant derived interleukin-1 beta (IL-1 beta) on the C4 column eluted with TFA-acetonitrile and the DVB column eluted with acetic acid-acetonitrile were similar, but only the polymeric column was able to separate the components present in an iodinated IL-1 beta preparation. Neither eluent had any harmful effect on the biological activity of IL-1 beta isolated after RP-HPLC. Several standard proteins could be separated when the polymeric column was eluted with acetic acid gradients in acetonitrile, isopropanol or water and, although the separation efficiency with acetic acid in water was lower than that in combination with classical organic modifiers, insulin, glucagon and human growth hormone (hGH) were eluted as sharp, symmetrical peaks. The recoveries of insulin and hGH were comparable for all three mobile phases (80-90%). The separation patterns obtained from a crude acetic acid extract of a normal and a diabetic, human pancreas analysed using acetic acid gradients with or without organic modifiers were found to be similar and comparable to those obtained on a silica C4 column eluted with an acetonitrile gradient in TFA. The principal differences resulted from the use of different UV wavelengths (215 nm for TFA-acetonitrile, 280 nm for acetic acid). Acetic acid extracts of recombinant derived hGH-producing Escherichia coli were separated on the DVB column eluted with an acetic acid gradient in water. Although the starting material was a highly complex mixture, the hGH isolated after this single-step purification was surprisingly pure (as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis). Consequently several (pure) polypeptides and complex biological samples were separated on a polymeric stationary phase eluted with acetic acid gradients in water without the use of organic modifiers.

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.

Analysis of proinsulin and its conversion products by reversed-phase high-performance liquid chromatography

Proinsulin is synthesized in the beta-cells of the endocrine pancreas, one of the four cell types found in the islets of Langerhans. Specific enzymatic cleavage of proinsulin results in the formation of equimolar amounts of insulin and C-peptide, via several intermediate split-proinsulin forms. Most mammals produce a single insulin, but in rodents two non-allelic insulin genes are expressed. There is an inverse ratio between the two insulins in rats and mice, the reason for this being unknown. It has been suggested that differences in transcription, translation (biosynthesis) and/or posttranslational processes (enzymatic conversion, intracellular degradation) could be possible explanations. Elevated amounts of proinsulin-immunoreactive material (PIM) have been described to occur in various conditions/diseases, suggesting alterations in beta-cell function, but the composition of the secreted PIM (intact proinsulin or its intermediates) has been incompletely determined. Studies of the biosynthesis of proinsulins and their conversion with the purpose of revealing some of these points depend on accessible reversed-phase high-performance liquid chromatographic (RP-HPLC) analyses capable of separating all the relevant, closely related polypeptides involved. This review will deal with the optimization of the RP-HPLC separations as well as sample preparation and recovery. Applications of the selected methods in the study of proinsulin biosynthesis and its conversion will also be presented.

High-performance liquid chromatography of rat and mouse islet polypeptides: potential risk of oxidation of methionine residues during sample preparation.

After preparative high-performance liquid chromatography of mouse islet culture medium, concentrated on disposable C18 cartridges (Sep-Pak), an unexpected insulin immunoreactive peak eluting earlier than mouse insulin I and II was detected. Molecular mass determination by mass spectrometry supported its suspected identity as methionine sulphoxide insulin II. We have examined the formation of Met-O derivatives of insulin II, glucagon and pancreatic polypeptide during sample preparation (Sep-Pak and Speed-Vac concentrating). The oxidation of methionine residues was found to depend very much on the buffer, the organic modifier and the procedure. In particular the use of methanol-trifluoroacetic acid resulted in extensive oxidation. The oxidation could be minimized by adding 2 mM dithiothreitol to the buffer and by degassing and/or nitrogen-bubbling of the buffer. Minimal formation of Met-O derivatives is important for the quantitation of methionine-containing polypeptides.