Electrochemical modifications of proteins. 1. Glycitolation.

When an electrochemical method is tentatively applied for N-alkylation of proteins, some Maillard reactions occur on the counter electrode. A low level of reductive N-alkylation of casein was obtained on a cathodic electrode, but the method unfortunately remains poorly efficacious in comparison with those using a hydride donor such as sodium cyanoborohydride. Electroassisted reductive N-alkylation is suitable only for basic proteins such as histones or lysozyme. For other proteins, an alternative consists of either methylating carboxylic residues to increase the value of their isoelectric pH or coating them with a cationic detergent.

The quantification of protein amino groups by the trinitrobenzenesulfonic acid method: a reexamination.

Trinitrobenzenesulfonic acid (TNBSA) is the reagent in a well-known method for quantification of primary amino groups, but even at room temperature, N-trinitrophenylation of primary amines is concomitant with hydrolysis of the reagent. The production of picric acid, the product of TNBSA hydrolysis, lowers the sensitivity of the method. Heating accelerates hydrolysis more than condensation on amino groups. The optimal pH range is centered on the value 10. The optical density is generally evaluated at 340 or 420 nm. The former value corresponds to the maximal absorption of the final product, N-trinitrophenylamines. The latter value is relative to the Mesenheimer pi-complex, the well-known intermediate of the overall reaction. Both wavelengths are suitable for quantification of amines, but 420 nm seems to be the best. Further addition of sulfite is not necessary. The quantification of amines is somewhat hampered by compounds such as area and sodium dodecyl anions. The relative rates of reaction of diaminoacyl groups of protein with TNBSA differ depending on the substitution degree of the neighboring groups. Some of them do not react. The trinitrophenylation kinetic seems to be more dependent on protein structure than reactivity. In evaluation of the available lysine in glycated proteins, the distinction between reductive alkylation and Maillard-type condensation necessitates quantitative evaluation of nonalkylated lysine in protein hydrolysate, compared to results with the TNBSA method. Our results are confirmed by complete guanidization of the protein and subsequent determination of homoarginine in hydrolysates.

Preparation of a dansylated fibrate, a new fluorescent tool to study peroxisome proliferation. Effect on hepatic-derived cell lines.

The synthesis of a dansylated fibrate (DNS-X) has been performed in order to identify the cellular affinity sites of peroxisome proliferators and to establish the subcellular localization of such molecules. DNS-X has been obtained by coupling the dansy1 chloride with the amine resulting from the bezafibrate alkaline hydrolysis. The purified DNS-X has been further characterized by spectrum analysis (UV-Vis, fluorescence, [1H]/[13C]-NMR and mass). At 250 microM and incubated for 48 h with the rat hepatic derived cells (Fao cells), DNS-X stimulates 12-fold the palmitoyl-CoA oxidase, a peroxisome proliferation marker enzyme. This increase is comparable to the one obtained with well known peroxisome proliferators such as bezafibrate or ciprofibrate. The stimulation by DNS-X is specific for the overall molecule since neither the dansyl chloride, the amine, nor the precursors of DNS-X are active. The increase of palmitoyl-CoA oxidase activity is correlated with the increase of the enzyme amount as shown by immunoblotting. In agreement with the species-specificity of the fibrate neither DNS-X, bezafibrate nor ciprofibrate significantly increase palmitoyl-CoA oxidase activity and the enzyme amount in human hepatic-derived cells, HepG2. This work shows that the dansylated fibrate is a new fluorescent tool to study the subcellular localization and identification of high affinity binding sites, then further on, to elucidate the peroxisome proliferation mechanism and the action of hypolipidaemic agents of the fibrate family.

Regio- and stereo-selectivity in the induction of peroxisome proliferation by substituted hexanoic acids.

Quantitative structure-activity relationship is an effective tool in order to predict drug potency. A similar approach is actually developed for peroxisome proliferation induced by substituted carboxylic acids issued from plasticizer metabolism in rats. The study is focused on acids found in rat urine after adipic diester dosings. Size, location of the substituted group and length of the chain have been studied. 3-D structure has also been taken in account for 2-ethyl hexanoic acids. The results obtained so far demonstrate that peroxisome proliferation potencies of the considered acids are modified according structure changes. At this time location of the group along the chain appears to be a predominant factor.

Enantioselectivity in the induction of peroxisome proliferation by 2-ethylhexanoic acid.

The stereoselectivity of the peroxisome proliferation potency of 2-ethylhexanoic acid (2-EHA), a metabolite of the plasticizer di-(2-ethylhexyl) adipate, was investigated in vitro. The enantiomers of 2-EHA were prepared via the semipreparative HPLC resolution of their diastereoisomeric (+)-(R)-1-phenylethylamine derivatives and the subsequent hydrolytic cleavage. Monolayers of hepatocytes were incubated 3 days with solution of (-)-(R), (+)-(S), and (+/-)-2-EHA. The peroxisome proliferation potency was measured by means of determination of the peroxisomal palmitoyl coenzyme A oxidation. The theoretical induction component due to each enantiomer were calculated from the experimental data considering the enantiomeric purities of the acids. The (+)-(S)-enantiomer was found to be the most potent inducer e.g., the eutomer, while the (-)-(R) was the distomer. The eudismic ratio was about 1.6 and the racemic mixture exhibited an intermediary potency. These results, obtained in vitro in conditions avoiding confounding factors such as pharmacokinetics, suggest that the peroxisome proliferation induced by 2-ethylhexanoic acid is a stereoselective phenomenon.