RESUMO
Ribonucleoside hydrolase C (RihC, EC 3.2.2.1-3.2.2.3, 3.2.2.7, 3.2.2.8) belongs to the family of ribonucleoside hydrolases that catalyze the cleavage of both purine and pyrimidine ribonucleosides to nitrogenous bases and ribose. Its most efficient reaction is the cleavage of uridine with the highest reaction rate. The reaction cannot be detected by a simple spectrophotometric method because of the same absorption maximum for the substrate and reaction product or requires time- and labor-consuming sample preparation for ribose. Reversed-phase HPLC is currently used to register enzymatic activity, where the time of one chromatographic run takes about 10 min. Since a large number of analyses is required to measure the kinetics of an enzymatic reaction, the total time is significant. In this work, we obtained new recombinant RihC from Limosilactobacillus reuteri by gene cloning and expression in E.coli cells. We proposed a new approach for determining the enzymatic activity of the new RihC using hydrophilic interaction liquid chromatography (HILIC). The novel column was developed for this procedure providing the determination of uracil and uridine with high efficiency and retention times of 0.9 and 1.7 min, respectively. Kinetic parameters for RihC uridine cleavage were determined. The proposed approach provided significant rapidity for measurement of the enzyme kinetics being 5 times faster as compared to reversed-phase HPLC.
Assuntos
Hidrolases de Éster Carboxílico , Ribonucleosídeos , Ribose , Ribonucleosídeos/análise , Cromatografia Líquida , Uridina , Escherichia coli/genética , Escherichia coli/metabolismo , Interações Hidrofóbicas e HidrofílicasRESUMO
A number of novel stationary phases for hydrophilic interaction liquid chromatography (HILIC) containing both amides and hydroxyl groups in the functional ligand were obtained by the multicomponent Ugi reaction on the aminated silica surface. The ligands were either attached directly to the substrate surface or spaced by 1,4-butanediol diglycidyl ether (1,4-BDDGE) as a linker. Chromatographic parameters of the obtained stationary phases were evaluated using Tanaka test and model mixtures of sugars, amino acids, and water-soluble vitamins. The adsorbent obtained by direct substrate modification by the Ugi reaction showed the increase of hydrophilicity according to the Tanaka test and high selectivity toward all the analytes with separation efficiency of up to 40000 N/m. The separation of 9 sugars in 12 min, 7 amino acids in less than 18 min, and 7 vitamins in less than 10 min with only 10 cm long column was provided. The effect of introducing 1,4-BDDGE as a linker between the substrate surface and the ligand formed via the Ugi reaction was demonstrated and discussed.
Assuntos
Compostos Orgânicos , Vitaminas , Cromatografia Líquida/métodos , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Dióxido de Silício/químicaRESUMO
Evaluation of the chromatographic properties of covalently bonded hyperbranched stationary phase based on poly(styrene-divinylbenzene) (PS-DVB) and containing zwitterionic fragments in the structure of functional layer was conducted in suppressed ion chromatography (IC), reversed phase high performance liquid chromatography (RP HPLC), and hydrophilic interaction liquid chromatography (HILIC) modes. Besides the possibility of resolving 20 inorganic anions and organic acids using KOH eluent in suppressed IC, prepared resin provided the separation of alkylbenzenes in RP HPLC, water-soluble vitamins, amino acids, and sugars in HILIC mode. Trends in the retention of hydrophobic and polar analytes on the prepared stationary phase indicated the dominating effect of analyte nature on the retention mechanism and proved satisfactory hydrophilization of PS-DVB surface with hyperbranched functional layer for retaining polar compounds. The obtained results revealed good prospects of using hydrophobic PS-DVB substrate for preparing stationary phases for mixed-mode chromatography.
