Biomedical subjects
Publications and source records attributed to H Zahn.
This paper is an autobiographical study of the author's early work on the chemical cross-linking of proteins as well as on oligomer and peptide synthesis from 1949 in Heidelberg until the synthesis of insulin in 1963 in Aachen.
This overview emphasizes present aspects of sulfur-containing amino acids in hair. A selection of analytical procedures to determine cystine, cysteine, S-sulfocysteine, cystine oxide, cysteic acid, lanthionine and lysinoalanine are presented. The methods relate to intact hair or partial and total hydrolysates and comprise chromatography, titration, colorimetry, polarography and spectroscopy. For the analysis of cysteine, cystine and cystine oxides, polarography and spectroscopy are the methods of choice. Cysteic acid, lanthionine and lysinoalanine are analysed by means of ion-exchange chromatography (Spackman et al., 1958) after total hydrolysis.
The alpha-glyceryl esters of Z-Gly, Z-Phe and Z-Tyr were synthesized and their use for protease catalyzed peptide synthesis was studied. Three enzymes isolated from crude papain were compared in their catalytic potency. Syntheses with alpha-chymotrypsin were performed in a biphasic system.
The chemical syntheses of C-terminally shortened analogues of C3a, which is the best investigated anaphylatoxin and derives from the third component of complement system, is reported. The peptide assembly was performed with the solid-phase technique using a polyamide support and an orthogonal protection strategy. The base-labile Fmoc group was chosen for N alpha protection in combination with acid-labile side-chain protection. Excellent acylation yields could be obtained using HBTU (O-benzotriazolyl-N,N,N',N'-tetramethyluronium hexafluorophosphate) as activating reagent. With this methodology we synthesized eighteen different peptides with the following modifications: Varying the peptide length by sequential addition of glycine or arginine residues, prolongating the N-terminus with the Fmoc- or Fmoc-aminohexanoyl residues and exchanging the glycine in position 74 for alanine or D-alanine. We obtained two C3a analogues, Fmoc-YRAAALALAR and Fmoc-Ahx-YRRGRAAALGLAR, which were shown to be substantially more active than native C3a in the guinea-pig-platelet assay.
A trifunctional reagent was designed which allows derivatization of ligands, particularly peptides and proteins, for subsequent photoaffinity labelling of receptors and specific isolation of the covalent complex or its fragments. B29-(2-nitro-4-azidophenyl)-biocytinyl-insulin (NB-insulin) was synthesized, radioiodinated, and the B26-mono-iodo derivative isolated by HPLC. It was used to photoaffinity label human placental membranes and the purified insulin receptor. Extensive digestion of the covalent insulin-receptor complex with trypsin (EC 3.4.21.4) led to the generation of a fragment of Mr 14,000. Specific complexing with avidin, derivatized avidin or streptavidin could be demonstrated for the photoaffinity labelled alpha-subunit and the 14,000 core fragment. The latter was isolated (approx. 100 pmol from 3-4 placentae) by streptavidin affinity chromatography and HPLC. According to microsequencing based on the known primary structure of the insulin receptor, the N-terminus of the core peptide appears to be Leu20-His21-Glu22-Leu23. We thus conclude: a part of the insulin-binding region of the receptor is located close to the N-terminus of its alpha-subunit in a remarkably stable domain of the sequence 20--(approx.) 120.
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After it has been shown that removal of residues B26-B30 leaves insulin with full biological activity, provided the new C-terminus is amidated (Fischer et al. (1985) Biol. Chem. Hoppe-Seyler 366, 521-525), it is demonstrated here that it does not even preclude enhancement of potency. 7 analogues of des-(B26-B30)-insulin-B25-amide were prepared by trypsin-mediated semisynthesis, the replacements being D-PheB24; HisB25, D-PheB25, TrpB25, TyrB25; D-PheB24,B25 and D-PheB24, TyrB25. Mere conversion of the configuration of B25-phenylalanine reduces in vitro potency to 0.5%. If B25-phenylalanine is, however, substituted by histidine or tyrosine activity is increased to 310 or 230, respectively. According to the features common to these two side chains, the favourable effect should be due to their ring structure with balanced aromatic and polar or H-bonding properties, respectively. The results indicate that in the complete insulin molecule the C-terminal pentapeptide modulates the subtle role that residues B24 and/or B25 play in receptor binding and activity; its presence may have a positive or negative effect. The drastic differences in activity between the shortened analogues are in no ways reflected in the CD spectra which are very similar, though clearly different from that of native insulin.
A simple preparation of Ac-Arg-(p-TosH or HCl)-NHMe is described. The NG-protonated Z-Arg was coupled with methylamine by the mixed anhydride method. Z-Arg-NHMe was purified as a NG-p-toluene sulfonate salt by crystallization from water. After removal of the Z group by catalytic hydrogenation and acetylation Ac-Arg(p-TosH)-NHMe was obtained. Ac-Arg(HCl)-NHMe was prepared by chromatography of the NG-TosH derivative on Dowex 44 (in Cl- form).
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The chemical synthesis and biological properties of N-(6-purinyl)peptides are described. N-(6-Purinyl)amino-acid derivatives were synthesized and condensed with amino acid esters and peptide esters using the dicyclohexylcarbodiimide/N-hydroxysuccinimide method. The products were isolated via gel filtration on Sephadex G-10 in 0.05M NH4HCO3 followed by either ion exchange chromatography on SP-Sephadex or by preparative HPLC. The methyl esters were saponified and the tert-butyl ester group was removed by treatment with trifluoroacetic acid without damaging the purinyl residue. N-(6-Purinyl)peptides were characterised by chromatographic and spectroscopic methods. Acid hydrolysis of N-(6-purinyl)-L-amino acids caused the racemization of the neighbouring L-amino acid. Model studies were performed with N-(6-purinyl)-L-alanine, N-(6-purinyl)-D-alanine, N-(6-purinyl)-L-alanyl-L-leucine and N-(6-purinyl)-D-alanyl-L-leucine. After acid hydrolysis the N-(6-purinyl)amino acids were totally racemized and the N-(6-purinyl)dipeptides formed 14% of the enantiomer of alanine. The N-(6-purinyl)-omega-amino acids and the N-(6-purinyl)peptides were screened in a limited number of tests as immunomodulators (antibody-secretion, phagocytosis, cytostatic activity of macrophages) and as cytotoxic agents.
Replacement of B25-phenylalanine by leucine in the insulin sequence causes marked inactivation. The effect of this sequence variation was studied here in des-(B26-30)-insulin. [LeuB25]des-(B26-30)-insulin and its B25-amide were prepared by trypsin-mediated semisynthesis from N-terminally protected des-(B23-30)-insulin and synthetic tripeptides. The relative lipogenic potency in isolated rat adipocytes was 8.0% for the truncated analogue with a free B25-carboxyl function, and 18.1% for the amidated analogue. Binding to cultured human IM-9 lymphocytes was 4% and 9%, respectively. Thus, both shortened insulins are markedly more active than [LeuB25]insulin. The PheB25----LeuB25 substitution in both the shortened and the full sequence has a moderate effect on the CD spectrum, indicating that the gross main chain conformation is largely retained in both molecules. Independent of the substitution an absolute increase of the circular dichroism is observed upon amidation of the B25-carboxyl group.
Des[(B26-30)-pentapeptide]insulin-B25-amide was prepared from protected des-[(B23-30)-octapeptide]insulin (pig) and H-Gly-Phe-Phe-NH2 by trypsin-mediated semisynthesis in a yield of 9% (based on insulin). The analogue was characterized with respect to chemistry, biological function and CD spectroscopy. While des[(B26-30)-pentapeptide]insulin with free carboxylate group exhibited a typical insulin activity of only 25% in vitro, des[(B26-30)-pentapeptide]insulinamide was fully active. Therefore des[(B26-30)-pentapeptide]insulin meets all structural and dynamic requirements for recognition and binding of the receptor as well as exertion of the biological effect, provided that the negative charge in the hydrophobic environment of PheB25 is neutralized.
A hexapeptide obtained from human casein by enzymatic digestion has been purified, sequenced and synthesized; its structure is: Val-Glu-Pro-Ile-Pro-Tyr. In vitro this hexapeptide stimulates the phagocytosis of opsonized sheep red blood cells by murine peritoneal macrophages. Administered intravenously to adult mice, it enhances the resistance to infection with Klebsiella pneumoniae.
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The chemical synthesis of [Tyr(I)A19] and [Tyr(I2)A19]insulin (porcine), using the amino-acid derivatives 3-iodotyrosine and 3,5-diiodotyrosine is described. The synthesis of the iodinated A-chains were performed by segment condensation in solution using acid labile protecting groups. The hydroxyl groups of Tyr(I) and Tyr(I2) were unprotected. For the temporary protection of the alpha-amino groups of the A-chain segments containing iodinated tyrosines, the 1-(4-biphenylyl)-1-methylethoxycarbonyl group was selected. After deprotection and sulphitolysis the iodinated A-chain tetra-S-sulphonates were purified by ion exchange chromatography on DEAE cellulose at pH 5.6. Reduction to the sulphhydryl form and the combination with native porcine B-chain yielded [Tyr(I)A19] and [Tyr(I2)A19]insulin (porcine), respectively. Purification of the first product was achieved by gel filtration and of the later by ion exchange chromatography on CM-cellulose at pH 4.5 and gel filtration. The monoiodinated insulin had a biological activity of 24 +/- 2% and the diiodinated analogue 2.6 +/- 0.2% as determined in an in vitro lipogenesis assay with epididymal adipocytes.
The chemical synthesis of two porcine insulin analogues is described. Leucine in position B17 of the native molecule was substituted by its D-enantiomer and by L-norleucine, respectively. Both B-chain derivatives were synthesized by fragment condensation and purified as di-S-sulphonates by gel filtration followed by ion exchange chromatography on SP-Sephadex at pH3. Combination with native sulphhydryl A-chain yielded [DLeuB17]insulin and [NleB17]insulin. Both insulin analogues were isolated by gel filtration followed by ion exchange chromatography on CM-cellulose at pH 4.0. Biological activities of the analogues were determined relative to native pork insulin: 1) glucose oxidation in rat epididymal adipocytes was 6% for [DLeuB17]insulin and 16% for [NleB17]insulin, 2) receptor-binding affinity tested with cultured human fibroblasts and with rat adipocytes was 3% for [DLeuB17]insulin and 26% for [NleB17]insulin, and 3) thymidine incorporation into DNA of human fibroblasts was 35% for [DLeuB17]insulin and 100% for [NleB17]insulin.
The yield of insulin by the combination of reduced A and B chains is increased from 15% to 33-34% if the non-insulin-containing fractions of the gel filtration are isolated and used for further combinations (recycling). Crude insulin recovered by gel filtration can be highly purified by preparative high-performance liquid chromatography. Lower yields of insulin were obtained on recombination of chains which had been recycled five times or more. Sulfur analyses show a loss of cystine sulfur. The occurrance of lanthionine and lysinoalanine indicates an alkaline destruction of cystine/cysteine during the repeated combination steps.
Four antisera raised against partly purified PTH preparations all showed a wide range of specificities when reacting with radioiodinated PTH peptides representing several different portions of the intact hormone sequence. In contrast, antisera raised against individual peptides were only able to cross-react with other peptides that contained all or part of their amino acid sequence in common. Cross-reacting peptides were seen to contain one or more amino acid residues having high interspecies variability in common. We have explained the antigenicity and cross-reactivity of the peptides on the basis of these common highly variable amino acid sequences. We have concluded that the selection of hormonal material in radioimmunoassays for PTH should be made on the basis of the highly variable amino acid residue content. This will allow a narrowing of the assay specificities and permit detection of a desired region of the PTH hormone.