Immobilisation of oligo-peptidic probes for microarray implementation: characterisation by FTIR, atomic force microscopy and 2D fluorescence.

Proteomic microarrays show a wide range of applications for the investigation of DNA-protein, enzyme-substrate as well as protein-protein interactions. Among many challenges to build a viable "protein microarray", the surface chemistry that will allow to immobilised various proteins to retain their biological activity is of paramount importance. Here we report a chemical functionalisation method allowing immobilisation of oligo-peptides onto silica surface (porous silica, glass, thermal silicon dioxide). Substrates were first derivatised with a monofunctional silane allowing the elaboration of dense and uniform monolayers in highly reproducible way. Prior to the oligo-peptides grafting, this organic layer was functionalised with an amino-polyethyleneglycol. The coupling step of oligo-peptides onto functionalised supports is achieved through activation of the C-terminal function of the oligo-peptides. Chemical surface modifications were followed by FTIR spectroscopy, AFM measurements and fluorescence scanning microscopy. A systematic study of the oligo-peptide grafting conditions (time, concentration, solvent) was carried out to optimise this step. The oligo-peptides grafting strategy implemented in this work ensure a covalent and oriented grafting of the oligo-peptides. This orientation is ensured through the use of fully protected peptide except the terminal primary amine. The immobilized peptides will be then deprotected before biological recognition. This strategy is crucial to retain the biological activity of thousands of oligo-probes assessed on a microarray.

Selective inhibition of rat heart cAMP phosphodiesterases by lipophilic C-methyl-2-phenyl-4H-1-benzopyran-4-ones (C-methylflavones).

A series of eight methoxylated C-methyl-2-phenyl-4H-1-benzopyran-4-ones 3, 6, 10-15 was evaluated as inhibitors of rat heart cytosolic cyclic nucleotide phosphodiesterase (PDE). The 2-(3,4-dimethoxyphenyl)-5,7-dimethoxy-3,8-dimethyl-4H-1-benzopyran-4-one (3) and the 2-(4-methoxyphenyl)-5,7-dimethoxy-3,8-dimethyl-4H-1-benzopyran-4-one (10) have never been previously described. Inhibition was performed on the whole cytosolic preparation and on the four PDE isoforms after HPLC purification. The flavones 3, 6, 10, 13 and 14 were selective and potent inhibitors of the isoforms, namely ROI (rolipram-sensitive) and CGI (cGMP-sensitive) PDEs specifically hydrolyzing cAMP. The di-C-methylflavones 3 and 13 have been shown to be potent inhibitors of these two isoforms, with IC50 values in the micromolar range.

Hydrolysis of alanine oligomers and of elastin by P. aeruginosa proteinases and thermolysin.

The hydrolysis of alanine oligomers by P. aeruginosa proteinases, thermolysin and porcine pancreatic elastase was studied. The concentrations of substrates and cleavage products were determined using reverse phase high pressure liquid chromatography. Tetraalanine was the shortest oligomer for which we could demonstrate hydrolysis by all the proteinases, except for porcine pancreatic elastase which only significantly hydrolyzed peptides longer than hexaalanine. Porcine pancreatic elastase hydrolyzes hexaalanine at a single site, whereas the other enzymes may split it either into two trialanine molecules, or into di- and tetraalanine, the latter being further cleavable to dialanine. A kinetic model based on first-order kinetic rate constants is proposed and the individual constants determined. Although P. aeruginosa elastase and thermolysin are closely similar in structure, they have shown a marked difference in their hydrolysis of either elastin or tetraalanine. Elastolytic activity of thermolysin was higher than that of elastase but tetraalanine was hydrolyzed more slowly by thermolysin.

Elastolytic activity of Pseudomonas aeruginosa elastase.

Elastolysis of insoluble elastin by Pseudomonas aeruginosa elastase was found to be less specific (higher apparent Km value) but more active (higher activity) than with pancreatic elastase. Furthermore, pancreatic and P. aeruginosa elastases act synergistically during the initial stages of elastolysis. After extensive hydrolysis, the size distribution of digestion products was lower with P. aeruginosa than with pancreatic elastase. The higher extent of hydrolysis may be explained by the fact that, if pancreatic elastase needs at least six sub-sites for activity, P. aeruginosa elastase may hydrolyse tetrapeptides such as tetraalanine, or synthetic substrates such as furylacryloyltripeptides FA-X-Leu-Y, X and Y being Gly and/or Ala.