Bz-Arg-OH

Bz-Arg-OH

Real-Time Imaging of Protease Action on Substrates Covalently Immobilised to Polymer Supports.[Pubmed:19779571]

Adv Synth Catal. 2007 Jun;349(8-9):1321-1326.

We report for the first time single bead spatially resolved activity measurements of solid-phase biocatalytic systems followed in real-time. Trypsin cleavage of Bz-Arg-OH and subtilisin cleavage of Z-Gly-Gly-Leu-OH each liberate a free amino group on aminocoumarin covalently immobilised to PEGA(1900) beads [a co-polymer of poly(ethylene glycol) with molecular mass of 1900 cross-linked with acrylamide]. This restores fluorescence which is imaged in optical sections by two-photon microscopy. For trypsin cleavage, fluorescence is restricted initially to surface regions, with more than 1 hour needed before reaction is fully underway in the bead centre, presumably reflecting slow enzyme diffusion. In contrast, for subtilisin cleavage fluorescence develops throughout the bead more quickly.

Protease-catalyzed tripeptide (RGD) synthesis.[Pubmed:10689065]

Enzyme Microb Technol. 2000 Feb 1;26(2-4):108-114.

The tripeptide Bz-Arg-Gly-Asp(-OMe)-OH was synthesized by enzymatic method. Bz-Arg-Gly-OEt was synthesized by trypsin in ethanol containing 0.1 M Tris/HCl buffer (pH 8.0), and then H-Asp(-OMe)(2) was incorporated into the Bz-Arg-Gly-OEt using chymopapain in 0.25M CHES/NaOH buffer (pH = 9.0, EDTA 10 mM). The yield of Bz-Arg-Gly-OEt and Bz-Arg-Gly-Asp(-OMe)-OH were 80% and 70% using 1M Bz-Arg-OEt and 0.5M Bz-Arg-Gly-OEt, respectively. For Bz-Arg-Gly-OEt synthesis reaction at high concentrations of the substrates, the buffer content in ethanol was a key factor to determine the optimal reaction condition. In Bz-Arg-Gly-Asp(-OMe)-OH synthesis reaction, the yield was low in organic solvent due to various side products such as Bz-Arg-OH, Bz-Arg-Gly-OH, and Bz-Arg-Gly-Asp(-OMe)-Asp(-OMe)-OH, suggesting that chymopapain has a very broad substrate specificity of the S(1) site. The Bz-Arg-Gly-Asp(-OMe)-OH synthesis rate and its yield were dramatically elevated and the side reactions were reduced using only the CHES/NaOH buffer (pH = 9.0, EDTA 10 mM) as a reaction media. The final product Bz-Arg-Gly-Asp(-OMe)-OH was identified to be formed via C-terminal hydrolysis of Bz-Arg-Gly-Asp(-OMe)(2) after the nucleophile, H-Asp(-OMe)(2), was added.

Anhydrotrypsin: new features in ligand interactions revealed by affinity chromatography and thionine replacement.[Pubmed:16873]

J Biochem. 1977 Mar;81(3):647-56.

Anhydrotrypsin was isolated in high purity from the product of base elimination from phenylmethanesulfonyl-trypsin, by a single operation of affinity chromatography. The adsorbent used for the chromatography was an agarose derivative coupled with peptides containing C-terminal arginine residues. As the affinity of the adsorbent for anhydrotrypsin was high compared with that for trypsin, purification of the enzyme derivative was easily achieved without the prior inactivation of trypsin which had been regenerated during the elimination reaction. Comparative studies of the ligand interaction specificities with anhydrotrypsin and trypsin confirmed the stronger interaction of the former protein with product-type ligands such as Bz-Arg-OH. No marked differences were observed between them in affinities toward substrate-type ligands such as Bz-Arg-NH2. The higher affinity of anhydrotrypsin was found to be limited to product-type ligands of L-configuration, i.e., the protein displayed an ability to discriminate the L-ligand from its optical isomer. THE PKa value for the ionization form of anhydrotrypsin responsible for the interaction with Bz-Arg-OH was estimated to be 7.60+/-0907