Hair sulfur amino acid analysis.

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.

Introduction of new crosslinks into proteins.

Analysis of the crosslinks epsilon-(gamma-glutamyl) lysine and epsilon- (beta-aspartyl) lysine present in treated wool has been improved by modifying the enzymic digestion. Treatment of wool with either monocarboxylic acid chlorides in dimethylsulfoxide or with 1-fluoro-2,4-dinitrobenzene in the presence of acetate considerably decreased epsilon-amino groups and solubility. Since no information of interchain amide crosslinks was observed, the hypothesis of so-called self-crosslinking postulated by ZAHN has to be withdrawn. The effects of both treatments are explained in the light of new results. The reaction of wool with glutaraldehyde leads to a stabilization of the fiber. Experiments with glutaraldehyde and primary alkyl amines as model componds revealed that the cyclic form of the aldehyde gave the unstable N-alkyl-2,6-dihydroxypiperidine, which either looses water to give N-alkyldihydropyridine or condenses with 2,6-dihydroxytetrahydropyran to yield a copolyether which was isolated. According to recent publications, crosslinking of proteins by glutaraldehyde is due to the formation of quaternary pyridinium compounds.

Progress report on hair keratin research.

Evidence is given for the progress in hair keratin research by bringing out four examples from the recent hair science literature. 1 Kon et al. [1] published a new method of solubilization and fractionation of the matrix and the microfibril proteins and found a significant decrease in the 'intact' microfibril keratin at the tip end of permed hair. 2 Contrary to the previous view of the existence of four major and one minor hair keratin pair, Langbein et al. [2, 3] showed that there are nine type I hair keratins and six type II hair keratins and drew up a two-dimensional catalogue of human hair keratins. 3 To collectively describe the extremely complex expression pattern of human type I and type II hair keratins in the hair follicle, Langbein et al. [2, 3] have summarized the corresponding RNA expression profiles of the various hair keratins schematically. 4 Contrary to the previous assignment of keratins exclusively to the microfibrils in the cortex, the mRNA expression studies of Langbein et al. [2, 3] implied that any hair cuticle cell leaving the living cell compartment contains four different hair keratins.

My journey from wool research to insulin.

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.

A study of the interaction of sodium dodecyl sulphate with the proteins of human heel stratum corneum.

Synopsis The aim of this research is to study the in vitro interaction of the anionic surfactant sodium dodecyl sulphate with human heel callus with special regard to the callus proteins. The sorption of SDS by callus is analysed at different pH values, demonstrating the expected maximum binding of the anionic surfactant at low pH. In both surfactant and blank solutions, swelling of the callus pieces occurs but to different extents. The sorption of SDS by callus is accompanied by a loss of free amino acids and proteins. The protein composition of the callus residues after chemical treatment and of the corresponding treatment baths is examined by amino acid analysis and shows differences in the respective molar amino acid ratios. Results obtained with more specific techniques, such as gel electrophoresis and immunoblotting, demonstrate identical as well as different protein components in the treatment baths depending on the experimental conditions (pH, blank or SDS). Although effects due to the surfactant treatment are in principle more distinct than with blank experiments, those of the latter cannot be neglected.

Fractionation of the chymotryptic precipitate of Bombyx mori silk fibroin.

1. A solution of Bombyx mori silk fibroin was digested with chymotrypsin. Amino acid analyses of the chymotryptic precipitate showed in addition to the main constituents Gly, Ala, Ser and Tyr, very small amounts of Lys, His, Arg, Asp, Thr, Glu, Pro, Cys, Val, Met, Ile, Leu, Phe and Trp. 2. A stable solution of the chymotryptic precipitate in 6m-urea was obtained by dialysing a solution in 50% (w/v) lithium thiocyanate against 6m-urea. 3. The dinitrophenylated chymotryptic precipitate in 6m-urea was fractionated by gel filtration and by ion-exchange chromatography. On Dowex 1 (X2), a main fraction I(d) and three further fractions with different amino acid compositions and molecular weights were obtained. 4. Specific rearrangement and fission of the bonds involving the serine nitrogen atoms of fraction I(d) and fractionation of the resulting mixture by gel filtration yielded five fractions. Two of these fractions had the compositions DNP-Ser-(Gly(6),Ala(4),Ser) and DNP-Ser-(Gly(4),Ala(2) or Ala(3),Ser) and are presumably double repeating units according to the proposed formula of Lucas, Shaw & Smith (1957), namely [Ser-Gly-(Ala-Gly)(n)](2), for n values of 2 and 1 respectively.

[Insulin analogues with permuted A chain N-terminus (author's transl)]. / Insulin-Analoga mit permutiertem N-Terminus der A-Kette

By partial synthesis insulin analogues were prepared in which the amino acid in position 1 of the A chain was permuted. Glycine in position A 1 was exchanged for leucine, tert.- butyloxycarbonylvaline, valine, proline, lysine as well as glutamic acid. Two pathways of partial synthesis were followed: Firstly, des-1-glycine-A-chain S-sulfonate was reacted with active esters of tert.-butyloxycarbonylamino acids. The ensuing modified A-chains were combined with natural B-chain to give A1-permuted insulins. In the second procedure, the preparation of tris-Boc-[A1-leucine]insulin was accomplished by reaction of Boc-leucine N-hydroxysuccinimide ester with NalphaB1,NepsilonB29-bis(tert.-butyloxycarbonyl)-des-A1-glycine-insulin. The protected insulin derivative had been prepared by combination of des-glycine-A-chain with Nalpha1,Nepsilon29-bis(tert.-butyloxycarbonyl)-B-chain. The deprotected analogues differed considerably in their CD-spectra from insulin and possessed low in vitro biological activities of 2.5-17%. Crystallization attempts failed. Thus, the introduction of side chains in position A1 distorts the conformation sterically and decreases the biological activity.

Contributions to the chemistry of human hair: II. Lipid chemical aspects of permanently waved hair.

Synopsis The cell membrane complex (CMC) and the internal lipids of untreated and permanent waved hair were investigated. The permanent waving procedure results in a lower amount of the cell membrane fraction that could be isolated compared to untreated hair. There is no difference in the amino acid composition of the CMC fractions. The internal hair lipids were extracted from the CMC fraction with chloroform/methanol. For untreated human hair the cell membrane complex yields 57% of lipid material, whereas from the CMC of permanent wave hair only 35% of lipids could be extracted. The polar lipids and the fatty acids are the main components of the internal hair lipids. After a permanent waving treatment there is a distinct decrease in the polar lipid fraction which is higher at alkaline (pH 9) than at neutral pH (pH 7). The reduction treatment at pH 9.0 also causes a cleavage of the fatty acid fraction in the internal hair lipids.

Contributions to the chemistry of human hair: III. Protein chemical aspects of permanent waving treatments.

Synopsis The influence of permanent waving on hair proteins was studied in order to obtain additional information about the chemistry of this cosmetic treatment. It was shown by amino acid analysis that with increasing reduction time during treatment fewer disulphide bonds were reformed in hair during subsequent reoxidation. Simultaneously, an increasing amount of sulphur-containing material is liberated from the hair, as demonstrated by the sulphur balance calculated from the sulphur-containing amino acids. The amount liberated is increased when an extensive soaking of the samples in water between the reduction and reoxidation step is performed. Comparing treatments with the use of reducing solutions of pH values between 7.5 and 10.0, it was found that the largest amount of cystine cleavage occurs at pH 9.0. All hair samples reduced at pH values above pH 8.5 showed incomplete reformation of the disulphide bonds during subsequent reoxidation. This was indicated by the content of free SH-groups and cysteic acid, as quantified by amino acid analysis. The damage to the hair proteins due to permanent waving was further confirmed by the determination of the pronase solubility. The reductive treatment of hair at pH 7.5 led to a relatively low degree of reduction, however all sulphur bonds were reformed during subsequent reoxidation.

Contributions to the chemistry of human hair. 1. Analyses of cystine, cysteine and cystine oxides in untreated human hair.

Synopsis Amino acid analysis, photometry and polarography were selected as analytical methods for the determination of thiol and disulphide groups in untreated human hair and the results are discussed. The pre-treatments necessary for the different analytical methods, e.g., hydrolysis, to some extent already induce chemical changes of the cysteine and cystine derivatives leading to method-dependent differences in the results. In many cases the partial oxidation products of cystine are supposed to be responsible for this effect. Electron Spectroscopy for Chemical Analysis (ESCA), as an analytical method for the determination of the cystine oxides, was found to be inapplicable due to insufficient resolution and sensitivity, whereas by the use of Fourier Transform Infrared (FTIR) Spectroscopy the sulphur-oxygen vibrations could be analysed and cystine monoxide and cystine dioxide were detected.

Use of Z-amino acid-glyceryl esters in protease catalyzed peptide synthesis.

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.

Structure-function relationships of des-(B26-B30)-insulin.

In order to study the role of the amino acid in position B25 and its environment in shortened insulins, a series of analogues was prepared with the following modifications: 1, Stepwise shortening of the B-chain including replacements of TyrB26 and ThrB27 by glycine; 2, substitutions at the carboxamide nitrogen of des-(B26-B30)-insulin-B25-amide by apolar, polar or charged residues of various chain lengths; 3, replacement of PheB25 by asparagine-amide, phenylalaninol or a series of alkyl and aralkyl residues. Trypsin-catalyzed semisyntheses were performed with Boc-protected or unprotected des-octapeptide-(B23-B30)-insulin and synthetic peptides. Relative receptor binding and in vitro bioactivity of [AsnB25]-des-(B26-B30)-insulin-B25-amide was 227 and 292% (on insulin), other activities ranged between 1 and ca. 200%. We make the following conclusions. An L-amino acid is essential in position B25. The B25-carbonyl and NH groups favour high binding and "superpotency", but are not indispensible for receptor contacts. For high affinity receptor interaction, the planarity at the C gamma-atom and the distance of B25-side-chain branching in position B25 are important, but an aromatic ring is not necessary.

[Synthesis of the fragments A1-,, A9-15 and A16-21 of ovine insulin A chain using the S-tert-butylmercapto residue for thiol protection (author's transl)]. / Synthese der Fragmente A1-8, A9-15 und A16-21 der Schafinsulin-A-Kette unter Verwendung des S-tert-Butylmercaptorestes als Thiolschutzgruppe.

The following paper describes the synthesis of the fragments A1-8, A9-15 and A16-21 of the ovine insulin A chain using the S-tert-butylmercapto residue for thiol protection. The synthesized fragments, which showed a good solubility in organic solvents, were partially deprotected with trifluoracetic acid and converted to the corresponding S-sulfonates quantitatively by oxidative sulfitolyses at pH 7.6.

The synthesis and some biological properties of N-(6-purinyl)peptides.

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.