Insulin B chain

Another solid phase fragment condensation with CDI and 1-hydroxybenzotriazole in the synthesis of the human insulin B-chain afforded the oligopeptide in 75% yield. The reaction time with the coupling pair CDI/HOBt was shorter than in the case of the DCC/ HOBt system.136 The CDI/HOBt activation method was also applied to the synthesis of a... 

C. A. Degradation of oxidized insulin B chain by the multiproteinase complex macropain (proteasome). Biochemistiy 1991, 30, 2725—2734. 

Desiderio, D.M. Katakuse, I. FAB-MS of Insulin, Insulin A-Chain, Insulin B-Chain, and Glucagon. Biomed. Mass Spectrom. 1984,11, 55-59. 

An octapeptide, corresponding in sequence to the C-terminal octapeptide found in the human insulin B-chain, is prepared by chemical synthesis. This octapeptide is then coupled to the shortened insulin using a well-established chemical method, thus yielding a semi-synthetic human insulin product ( human insulin emp Box 8.2). 

The first pyrolysin to be cloned from a higher plant was cucumisin from Cucumis melo, an extracellular protease highly abundant in melon fruit [152], Cucumisin was shown to have a broad substrate specificity in that it cleaves a variety of small peptide substrates and eight peptide bonds within the oxidized insulin B chain [153-155]. A similar, broad... 

FIGURE 10.22 Electropherograms of the products following on-chip tryptic digestion of bovine insulin B-chain at 37°C. The flow in the reaction channel was stopped for different times to study the effect on the reaction. Control runs (see the flat curves) without insulin B-chain were performed in the continuous flow mode and in a flow with a stop time of 6 min. The arrows indicate the migration time of benzylamine, which was added as an EOF marker. All electropherograms are plotted on the same scale with an offset for clarity [1058]. Reprinted with permission from Elsevier Science. 

Other name Aspergillus sojae neutral proteinase II Microbial neutral proteinase II Preferential cleavage of bonds with hydrophobic residues in Pi also Asn3- -Gln and Glys-J.-Ser bonds in insulin B chain. For calf thymus histone H4, high activity towards... 

Hie His-L bond of the- insulin B chain is cleaved within the senuence but not in the sequence Gln4-His3-Leu6-Cys7, because of the negative charge in the P2 position. Likewise, the Val-Glu bond is left intact... 

FIGURE 13 Plot of the logarithm of the retention volume (In VR) versus the concentration of the displacing salt, ammonium sulphate, in the HP-HIC mode with the proteins I, insulin B-chain 2, bovine trypsin inhibitor 3, bovine trypsinogen 4, insulin A-chain 5, ribonuclease 6, sperm whale myoglobin 7, horse heart cytochrome c. Data from Ref. 42. 

Fig. 5. The separation of polypeptide standards on Hypersil ODS with 0.1 M NaHjP04-H3P04, pH 2.1, as the mobile phase, at a flow rate of 1 ml/min. The peaks are as follows 1, Trp 2, lysine vasopressin 3, arginine vasopressin 4, oxytocin, 5, ACTHj-j, 6, insulin A-chain 7, bombesin 8, substance P 9, somatostatin 10, insulin B-chain 11, human calcitonin 12, glucagon 13, salmon calcitonin 14, melittin. Adapted from Fig. I of O Hare and Nice (1979).

Semisynthesis has become a suitable technique for chemical variations of large polypeptides or proteins from natural sources. Human insulin differs from porcine insulin only in position B30, where alanine is the C-terminal B-chain residue of porcine insulin but threonine is the C-terminal of the human insulin B-chain. Among the various ways to obtain human insulin from porcine insulin by protease-catalyzed semisynthesis, the one-step conversion by exchange of the C-terminal alanine B of porcine insulin with a threonine residue seems to be the method of choice. Several proteases, e.g. trypsin, achromobacter protease, and car-boxypeptidase Y, are able to catalyze the conversion and various groups have independently described an essential improvement of the enzymatic semisynthesis of insulin.The Hoechst procedure,which was developed as an industrial process, is described below as an example of a large-scale conversion of porcine insulin to human insulin in kilogram amounts for therapeutic application. Based on this type of transamidation reaction, it is easy to prepare various B30-insulin analogues. 

A. Lane 1, Molecular weight markers lane 2, Purified final product. Molecular mass markers are Novex SeeBlue Pre-Stained Standards and range as follows (from top to bottom) Myosin, 250-kDa BSA, 98-kDa Glutamic dehydrogenase, 64-kDa Alcohol dehydrogenase, 50-kDa Carbonic anhydrase, 36-kDa Myoglobin, 30-kDa Lysozyme, 16-kDa Aprotinin, 6-kDa Insulin B chain, 4 kDa. 

Figure 3 The structural levels of proteins, exemplified by human insulin in the T6 form. (A) Primary structure residues 15-18 of human insulin B-chain, shown as sticks. (B) Secondary structure residues 8-20 of the B-chain form an a-helix, here depicted as a superposition of sticks, and a cartoon-representation. (C) Tertiary structure insulin A- and B-chains fold up to a monomer, which is assumed to be the active form, binding to the insulin receptor. Insulin can exist in different oligomeric forms, depending on formulation and protein concentration. (D) The Zn -stabilized hexamer form is shown. 2 Zn ions are bound per insulin hexamer (only one Zn2" "-ion is visible in this view). The hexamer is a trimer of dimmers. Figure based on pdb-file IMSO, produced in Pymol. Source Bente Vestergaard, Biostructural Research, Faculty of Pharmaceutical Sciences, University of Copenhagen.

Insulin lispro (rDNA origin) is Lys(B28), Pro(B29) human insulin analog, created when the amino acids at positions 28 and 29 on the insulin B-chain are reversed. It has the empirical formula C257H383N65077S6 and a MW of 5808, both identical to that of human insulin. 

R8. Rinderknecht, E., and Humbel, R. E., Amino-terminal sequences of two polypeptides from human serum with non-suppressible insulin-like and cell-growth-promoting activities Evidence for structural homology with insulin B chain. Proc. Nad. Acad. Sci. 

Larsen et al. reported the enzymatic cleavage of a desB30 insulin B-chain from a presequence (Lys(Boc))6. This spacer shifts the conformation of the growing peptide chain from a p-structure to a random coil conformation and reduces peptide-chain aggregation, which otherwise causes serious synthetic problems. Novasyn KA- 305 was employed as a solid support, but unfortunately, no information about the enzyme used was reported 306. ... 

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