Development of a Capillary Electrophoresis Assay Based on Free Sulfate Determination for the Direct Monitoring of Sulfoesterase Activity

International audienc

Inhibition by heparin and derivatized dextrans of Staphylococcus aureus adhesion to fibronectin-coated biomaterials

Recent data on cardiovascular device-centered infections suggest that some plasma and extracellular matrix proteins contribute to bacterial adhesion and colonization on biomaterials. We previously developed an in vitro assay to study the Staphylococcus aureus adhesion-promoting effect of surface-adsorbed fibronectin on flat PMMA coverslips coated with a monolayer amount of fibronectin. We screened the potential anti-adhesive properties of a group of substituted dextrans, previously shown to exhibit potent anticoagulant and anticomplementary activities. In comparison to unsubstituted dextran which showed no significant (< 20%) adhesion inhibition at 1 mg/ml, dextrans increasingly substituted with carboxylic and benzylamide groups (CMBD) exhibited increasing anti-adhesive activities. Three CMBD derivatives showing an increasing proportion (5-14%) of benzylamide groups showed inhibition of bacterial adhesion increasing from 33 to 51% at 1 mg/ml. Another category of substituted dextrans having a variable proportion (2-26%) of sulfonated benzylamide groups (CMBDS) produced active inhibition of S. aureus adhesion. In comparison to these heparin-like dextran derivatives, native heparin produced inhibition values of S. aureus adhesion which were intermediate between those of CMBD and CMBDS compounds. Furthermore, the anti-adhesive activity was still expressed when substituted dextrans were preincubated with fibronectin-coated PMMA but washed away at the time when radiolabeled bacteria were added to the adhesion assay. This indicates that the anti-adhesive effects of CMBDS could be exerted at the level of the S. aureus binding site of fibronectin. In conclusion, S. aureus adhesion on fibronectin-coated biomaterials can be efficiently blocked in vitro by soluble compounds such as dextran derivatives

Specific adsorption of serine proteases on coated silica beads substituted with amidine derivatives

Amidine derivatives interact with serine proteases, the inhibition being due to interactions between amidine functions and the active sites of the enzymes. Five differents type of amidine (substituted or unsubstituted) were coupled to coated silica beads, which had previously been coated with DEAE-dextran to minimize the non-specific interactions due to silanol groups. Coated silica functionalized with substituted amidines shows a strong affinity towards human plasmin. This affinity is probably due to hydrophobic interactions between the substituted amidine and the human plasmin structure. Coated silica grafted by p-aminobenzamide gives a specific interaction with human plasmin. The importance of ionic strength and the steric conformation of the ligand is discussed. This support was used to purify thrombin from crude preparations by high-performance affinity chromatography. © 1990.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Affinity of human thrombin for insoluble polystyrene substituted with sulfonate, n‐hydroxyethylsulfamoyl and n‐substituted amidine functions

A new polymeric material was prepared from polystyrene beads with N‐substituted amidines and rendered hydrophilic with sulfonate and N‐hydroxyethylsulfamoyl groups. It was synthesized by reacting the p‐chlorosulfonyl polystyrene with ethanolamine followed by the Michael‐like addition of the terminal OH of the polymer (PSEa) to N‐t‐butyl‐N'‐phenylpropenamidine 1 thus leading to the final polymer PSEaAm. Its affinity constant for human α‐thrombin calculated from the Langmuir adsorption isotherms was about 106 M−1, a value comparable to the one estimated for polystyrene modified with L‐arginine methyl ester. This new material was used as stationary phase in affinity chromatography of thrombin confirming a strong and specific interaction between the positively charged amidinium groups and the enzyme at pH = 7.4 and desorption at pH = 10 and high ionic strength. Copyright © 1988 Hüthig & Wepf VerlagSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Temperature-responsive size-exclusion chromatography using poly(N-isopropylacrylamide) grafted silica

Silica-based packing materials induce non-specific interactions with proteins in aqueous media because of the nature of their surface, mainly silanol groups. Therefore, the silica surface has to be modified in order to be used as stationary phase for the High Performance Size-Exclusion Chromatography (HPSEC) of proteins. For this purpose, porous silica beads were coated with hydrophilic polymer gels (dextrans of different molecular weights) carrying a calculated amount of diethyl-aminoethyl groups (DEAE). Actually, as shown by HPSEC, these dextran modified supports minimize non-specific adsorption for proteins and pullulans in aqueous solution. Then, in order to change the pore size in response to temperature, temperature responsive polymer of poly(N-isopropylacrylamide) (PIPAAm) was introduced into the surface of dextran-DEAE on porous silica beads. The structure of these supports before and after modification was alternately studied by Scanning Electronic Microscopy (SEM) and Scanning Force Microscopy (SFM). An adsorption of radiolabelled albumin was performed to complete our study. Silica modifications by dextran-DEAE and PIPAAm improve the neutrality of the support and minimize the non-specific interactions between the solid support and proteins in solution. At low temperature, the support having PIPAAm exhibits a high resolution domain in HPSEC and finally permits a better resolution of proteins and pullulans. At higher temperature, hydrophobic properties of PIPAAm produce interactions with some proteins and trigger off a slight delay of their elution time