Aminopropyl derivatized Perloza beaded cellulose was acylated with alpha-bromoacetic anhydride to give alpha-bromo-acetamidopropyl Perloza. (N-Acetyl)-Cys-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2, the 7 C-terminal amino acids of the decapeptide luteinizing hormone-releasing hormone with a cysteine added to the N-terminus, was synthesized using Fmoc chemistry. The purified peptide (1.35-1.9 eq) was coupled to alpha-bromoacetamidopropyl Perloza in 0.1 M NaHCO3 solution, pH 8.3, for 1-2 hours. The peptide was anchored to the support via a thioether linkage. Analysis of the peptide-Perloza conjugate indicated near-quantitative displacement of support-bound bromine by the peptide. The peptidic affinity matrix was able to bind ovine antibodies to luteinizing hormone-releasing hormone (LHRH). Thioether immobilization offers directed, chemically stable, high-yield anchoring of synthetic peptides onto a chromatographic support. The high reaction efficiency means there is little waste of valuable synthetic peptide.

Acetates↗

PubMedDate not supplied

Side-chain deprotection of peptides synthesized onto a beaded cellulose support.

Peptide-Perloza beaded cellulose conjugates were synthesized. However, the concentrated TFA solutions usually used for deprotecting peptide-support conjugates were found to be unsuitable for use with Perloza because they destroyed the chromatographic flow properties of the matrix. Conditions for non-destructive (to the matrix) deprotection of the resin-bound peptide were determined in this study. A cocktail of m-cesol/EDT/thioanisole/TMSBr/TFA/DCM (1:1:2:2:15:79 by volume-Reagent D) was found to cleave amino acid side-chain-protecting groups while leaving the chromatographic properties of the peptide-resin unaltered. Although treating aminopropyl Perloza with solutions of TFA in DCM resulted in decrease of amine substitution, treating peptide-Perloza conjugates with identical reagents gave minimal loss of peptide.

Amino Acid Sequence↗

PubMedDate not supplied

Solid-phase synthesis of a peptide-ligand affinity matrix for isolation of chymosin.

Aminopropyl Perloza beaded cellulose was used as the support for solid-phase synthesis of resin-bound Val-dLeu-Pro-Phe-Phe-Val-dLeu, an inhibitor of aspartic proteases. Both Boc and Fmoc SPPS methodologies were employed in separate syntheses. The peptide-resins were characterized by amino acid analysis. The peptide-resin from the Fmoc synthesis gave the better amino acid analysis of the two syntheses and was used for further studies. Following modification of the peptide N-terminus by succinylation, the peptide-resin was able to bind chymosin (E.C. 3.5.21.4). The peptide resin was used for isolation of chymosin from a crude recombinant broth.

Amino Acid Sequence↗

PubMedDate not supplied

Glycosaminoglycan specificity of a heparin-binding peptide.

Glycosaminoglycans are complex sulfated polysaccharides with a diverse range of biological functions. Three glycosaminoglycan standards--chondroitin sulfate, dermatan sulfate and heparin--were characterized during this study. The interaction of the heparin binding site of protein C inhibitor, represented by the peptide sequence 264-283, in solution with the above glycosaminoglycan standards was studied. Circular dichroism spectroscopy was used to determine the dominant secondary structure induced in the peptide upon binding the relevant glycosaminoglycans. The various glycosaminoglycans induced different secondary structures. The level of induced secondary structure by dermatan sulfate and heparin was approximately twice that induced by chondroitin sulfate. For chondroitin sulfate and heparin, alpha-helix was the dominant ordered secondary structure, whereas for dermatan sulfate the beta-strand conformation dominated. The order of secondary structure induction of the protein C inhibitor peptide by the glycosaminoglycans paralleled the reported biological activities of these glycosaminoglycans for mediation of the biological activity in the intact protein. The strength of the interaction of dermatan sulfate and heparin with the protein C inhibitor peptide was measured by determining the concentration of salt required to inhibit 50% of the interaction. The values determined were 0.1 and 0.3 M salt for dermatan sulfate and heparin, respectively. These results show that different glycosaminoglycans can support different secondary structures in the protein C inhibitor peptide.

Amino Acid Sequence↗