The hexa- and pentapeptide extension of proalbumin. II. Processing of specific antibodies against the synthetic hexapeptide.

The chemically synthesized proalbumin hexapeptide was coupled to rabbit albumin with carbodiimide. Subsequently rabbits were immunized by subcutaneous injection of the conjugate. After 5 weeks the rabbits had developed antibodies against the hexapeptide, which could be detected by immunodiffusion. A sensitive enzyme-linked immunosorbent assay was developed. Using the hexapeptide and other very similar peptides as antigens, a high specificity of the antibodies against the proalbumin hexapeptide was found.

The hexa- and pentapeptide extension of proalbumin. I. Chemical synthesis of serum albumin propeptides.

The proalbumin hexapeptide extension was synthesized beginning from the C-terminal end by stepwise N-terminal peptide chain elongation starting from N-tert-butyloxycarbonyl-(Ng-nitro)arginyl-(Ng-nitro)arginine 4-nitrobenzyl ester; [alpha](20)365-12 degrees C (c = 1; dimethylformamide). The other amino acids were incorporated by excess mixed anhydrides of Ddz-amino acids (Ddz; 3,5-dimethoxyphenylisopropyloxycarbonyl) yielding the fully protected hexapeptide in crystalline quality. After removal of the protective groups by acid treatment and hydrogenation the peptide was purified by Dowex ion-exchange and Sephadex chromatography. The purity was confirmed by thin-layer chromatography and amino acid analysis.

Measurement of bactericidal/permeability-increasing protein in human body fluids by sandwich ELISA.

A sensitive sandwich ELISA has been developed to measure levels of native bactericidal/permeability-increasing protein (BPI) as well as two recombinant forms of BPI (rBPI and rBPI23) in human body fluids. The linear range for the rBPI and rBPI23 standard curves were 100-6000 pg/ml and 25-800 pg/ml respectively. Recovery of different concentrations of rBPI spiked into pooled human plasma samples averaged 83% and ranged from 65% at 300 ng/ml to 97% at 3 ng/ml. Recovery of rBPI23 averaged 56% and ranged from 30% at 0.5 ng/ml to 90% at 50,000 ng/ml. Because LBP is present in normal human plasma and shares sequence homology with BPI, the effects of rLBP on the BPI ELISA were also evaluated. Under standard assay conditions, rLBP caused minimal interference with BPI detection. At 100 micrograms/ml, rLBP generated a signal equivalent to 3 ng/ml of rBPI and 0.6 ng/ml of rBPI23. Matched serum and plasma samples were collected from 20 healthy adults to measure endogenous levels of BPI. The range of BPI concentrations was < 0.2-2.1 ng/ml in plasma and 4.9-72.1 ng/ml in serum. Western blot analysis indicated that the BPI ELISA immunoreactivity in plasma and serum correlated with the presence of a protein doublet (M(r) approximately 60,000), which comigrated with native BPI extracted from human neutrophils. These data demonstrate that low levels of holo-BPI are present in plasma, and suggest that additional quantities of BPI were released from neutrophils during the process of coagulation.

[Phagotest and Bursttest (Phagoburst), test kits for study of phagocyte functions]. / Phagotest und Bursttest (Phagoburst), Testkits zur Untersuchung von Phagozytenfunktionen.

Phagotest and Bursttest are complete test kits for the investigation of the phagocytic function of granulocytes and monocytes in whole blood using flow cytometry. Phagotest allows the quantitative determination of leukocyte phagocytosis (uptake of bacteria). It determines the percentage of phagocytes which ingest fluorescein-isothiocyanate (FITC) labelled opsonized E. coli bacteria and their activity (number of bacteria per cell). Reduced phagocytosis is observed in patients with sepsis, renal failure, and recurrent bacterial infections. Bursttest allows the quantitative determination of leukocyte oxidative burst. It determines the percentage of leukocytes which oxidize the fluorogenic substrate dihydrorhodamine (DHR) 123 and their enzymatic activity (amount of rhodamine 123). Reduced burst activity is observed in patients with chronic granulomatous disease (CGD).

Synthesis of the sequential polypeptide poly[Cys(Acm)-Ser-Phe-Glu-Glu] as a model for hydrolytic enzymes.

To construct a possible model of hydrolytic enzymes, the sequential polypeptide H[Cys(Acm)-Ser-Phe-Glu-Glu]nOH, n = 3-110, was prepared by polycondensation of H-Cys(Acm)-Ser(But)-Phe-Glu(OBut)-Glu(OBut)-O Su/1-hydroxybenzotriazole. In a solid state the polypeptide material showed beta-conformation both before and after cleavage of t-butyl protecting groups. The pentapeptide unit was synthetized by the Merrifield method utilizing modifications as follows: Gel phase synthesis on less than 0.5% cross-linked copolystyrene (quasi dissolved state). Centrifugal reactor. Ddz-amino acids, deprotected by 5% trifluoroacetic acid/dichloromethane/15 min. 3-Nitrophthalic anhydride to suppress formation of false sequences. Continuous photometric control of the completion of all operations during synthesis and transesterfication, yielding Ddz-Cys(Acm)-Ser(But)-Phe-Glu-(OBut)-Glu(OBut)-OMe (0.827 g; 53%, m.p. 180-182 degrees).

Synthetic insulin by selective disulfide briding, II. Polymer phase synthesis of the human B chain fragments.

Five protected fragments Ddz-Phe-Val-Asn(Mbh)-Gln(Mbh)-His(Dnp)-Leu-Cys(Acm)-Gly-OH [I], Ddz-Ser(But)-His(Dnp)-Leu-Val-Glu-(OBut)-Ala-OH [II], Ddz-Leu-Tyr(But)-Leu-Val-Cys(Mbzl)-Gly-OH [III], Ddz-Glu(OBut)-Arg(Tos)-Gly-OH [IV], Ddz-Phe-Phe-Tyr(But)-thr(But)-Pro-Lys(Z)-Thr(But)-OH [V] for the synthesis of the human insulin B chain, were prepared by an efficient procedure on solid phase.

Preparative merits of the mixed anhydride (MA) method in the excess use of DDZ-amino acids in the peptide synthesis of biologically active new antamanide analogues.

The mixed anhydride (MA) method of peptide synthesis is further simplified by the repetitive excess use of Ddz-amino acids. In six comparative preparations of decapeptides this is demonstrated by the ease and speed of the synthetic manipulations, the efficiency of the monitoring of the reactions and purifications, and by the complete recycling of excess amino acid derivatives. In using of Ddz-amino acyl isobutylformate mixed anhydrides, side reactions are effectively suppressed, as proved by good coupling of Ddz-proline on to prolyl-peptides. Hydrophilic, crystalline and biological active antamanide analogues are obtained containg various functional side groups. The known cis-conformation of the antamanide prolyl-prolyl bonds is also established in the analogues by 13C-n.m.r. measurements. All new compounds are characterized by molecular peaks in the mass spectra.

Analogs of amanin. Synthesis of Ile3-amaninamide and its diastereoisomeric (S)-sulfoxide.

Ile3-amaninamide (3-R) and its diastereomeric sulfoxide (3-S) are obtained by oxidation of the bicyclic thioether peptide 2 by hydrogen peroxide in acetic acid. 2 was prepared by an intramolecular Savige-Fontana reaction of the linear octapeptide tert.-butylester 4 whose N-terminal Boc-Hpi residue on treatment with TFA loses the Boc group and reacts under thioether formation with the released cysteine-SH. The concomitantly deprotected carboxyl terminus is coupled intramolecularly with the free amino group of the secocompound 5 using the MA or DCCI method, thus forming the homodetic peptide ring. Compounds 3-R and 3-S agree very well with analog samples in chiroptical behavior. Thioether 2 and sulfoxide 3-R exert 50% inhibition of RNA polymerase II (or B) from Drosophila melanogaster in 10(-6) M solution whereas Ki of 3-S is about five times higher.

Syntheses of monocyclic and bicyclic peptides of tryptathionine and glycine.

L-3a-Hydroxy-1.2.3.3a.8.8a-hexahydropyrrolo [2,3-b-]-indole-2-carboxylic acid (Hpi), obtained from L-tryptophan by oxidation with peroxyacetic acid (Savige, 1975), after introduction of the Boc-residue at N-1, is coupled with various glycine peptides of S-trityl-L-cysteine to give the Hpi-peptides 6(a-f). By treatment with absolute trifluoroacetic acid these peptides are converted by an intramolecular thiolysis to the monocyclic thioethers 7(a-f). Two of them, 7e and 7f, can be subjected to a second cyclization by the mixed anhydride method thus yielding the bicyclic tryptathionine heptapeptide 8e and octapeptide 8f. In their structures the bicyclic molecules resemble the mushroom phallotoxins and amatoxins, respectively. They show CD spectra closely related to the naturally occurring bicyclic peptides thus indicating conformational similarities. The CD spectra of the other cyclic peptides synthesized are also presented and discussed.

Carbon-13-N.M.R.-spectra of antamanide, several analogues and their alkali ion complexes.

Natural abundance carbon-13 Fourier transform n.m.r.-spectra were obtained of the cyclic decapeptides [Phe4Val6] antamanide (I); antamanide (II); [Tyr5] antamanide (III); [Ala1] antamanide (IV) and their ion complexes (I)-Na+, (II)-Na+, (II)-Li+, (III)-Na+ and (IV)-Na+. Based upon literature data, systematic comparisons and model compounds, a line assignment approach was performed for the majority of the aliphatic carbons. The spectra of the [Ala9Val6] analogues (V) (Bystrov et al., 1972) and II (measured in CD3CN Patel, 1973a, b) and their complexes were also assigned. Characteristic chemical shift variations observed upon complex formation were calculated (delta delta free peptide[Me+ complex) for a number of corresponding carbons, revealing shift changes up to 2.4 p.p.m. Preliminary calculations of the theta angle at selected prolines for some ion complexes are included.

Solid phase synthesis and biological activity of mast cell degranulating (MCD) peptide: a component of bee venom.

Mast cell degranulating (MCD) peptide, a 22 amino acid residue basic peptide from bee venom, was synthesized by stepwise solid phase synthesis on a benzhydrylamine resin support. N alpha-t-butyloxycarbonyl and benzyl type side chain protection was used. The two disulfide bridges were formed selectively by using S-acetamidomethyl protection for the cysteine residues in position 5 and 19 and S-methylbenzyl protection for the cysteine residues in positions 3 and 15. Crude synthetic MCD peptide was obtained following deprotection and cleavage from the resin by the low/high HF method. The peptide was isolated in pure form by ion exchange chromatography and gel filtration. The final product has physical, chemical, and biological properties identical with those reported for the natural product. The synthetic strategy utilized for MCD peptide will facilitate the availability of structurally similar analogs for evaluating antihistaminic and anti-inflammatory activities.

Biochemical characterization and immunomodulatory action of thymic components as determined by flow cytometry on human lymphocytes.

A commercial preparation from calf thymus was separated into its components, then chemically characterized and tested for immunomodulatory efficiency. For the latter, a novel in vitro test system is described as a relatively simple screening procedure. It employs whole blood cultures and flow cytometry with fluorescent monoclonal antibodies defining human T-cell subsets. As a reference substance, thymosin fraction 5 was used. It could be shown that the effects of the entire thymic preparation as well as of one of its peptidic components resembled closely the ones obtained with thymosin fraction 5. In several cases, i.e. with donors relatively deficient in suppressor/cytotoxic T-lymphocytes, the effect of the former was even superior to that of thymosin fraction 5.