title: Validation of a thin-layer chromatography/densitometry method for the characterization of invertase activity.
abstract: This paper presents a kinetic study of invertase, a specific fructofuranosidase cloned from the Leishmania major genome. The kinetic parameters of the β-d-fructofuranosidase from Leishmania major (BfrA) were determined using Thin-Layer Chromatography (TLC) and UV-densitometry (TLC@UV) specifically developed for the separation and detection of three carbohydrates namely sucrose, glucose and fructose. Separation was performed on TLC silica gel 60 F254 plates impregnated with sodium bisulphate and citrate and heated prior to development. This fast and easy separation was performed with two successive developments using ACN/H2O 80/20 (v/v) as mobile phase. Sensitive and repeatable derivatization of sugars was achieved by dipping the plates in a solution of 4-aminobenzoic acid. Quantification was performed by UV-detection. The method was validated according to ICH guidelines Q2(R1) in terms of specificity, limits of detection and quantification, precision and robustness (with n=3 replicates and CV ≤10%). The characterization of BfrA reaction kinetic was performed by monitoring the accumulation of either glucose or fructose detected by TLC@UV. Hydrolysis of sucrose was described by the Michaelis-Menten kinetic parameters (KM; Vmax) respectively equal to 63.09±7.590mM; 0.037±0.00094mM/min using glucose production and 83.01±14.39mM; 0.031±0.0021mM/min monitoring fructose. Hydrolyses of three alternative substrates, raffinose, stachyose and inulin, were also compared and the regiospecificity of the reaction was characterized. This TLC@UV method is shown to be suitable for the refined kinetic analysis of different reactions related to the hydrolysis of sugars.
authors.affiliation: Univ. Orléans, CNRS, ICOA, UMR 7311, F-45067 Orléans, France. Electronic address: justine.ferey@etu.univ-orleans.fr.
authors.forename: Justine
authors.initials: J
authors.lastname: Ferey
authors.affiliation: Univ. Orléans, CNRS, ICOA, UMR 7311, F-45067 Orléans, France.
authors.forename: David
authors.initials: D
authors.lastname: Da Silva
authors.affiliation: Univ. Orléans, CNRS, ICOA, UMR 7311, F-45067 Orléans, France.
authors.forename: Sophie
authors.initials: S
authors.lastname: Bravo-Veyrat
authors.affiliation: Univ. Orléans, CNRS, ICOA, UMR 7311, F-45067 Orléans, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Lafite
authors.affiliation: Univ. Orléans, CNRS, ICOA, UMR 7311, F-45067 Orléans, France.
authors.forename: Richard
authors.initials: R
authors.lastname: Daniellou
authors.affiliation: Univ. Orléans, CNRS, ICOA, UMR 7311, F-45067 Orléans, France.
authors.forename: Benoît
authors.initials: B
authors.lastname: Maunit
mesh_terms: D002138:Calibration; D002855:Chromatography, Thin Layer / Q000379:methods*; D003720:Densitometry / Q000379:methods*; D005632:Fructose / Q000032:analysis; D005947:Glucose / Q000032:analysis; D007700:Kinetics; D018320:Leishmania major / Q000201:enzymology; D012015:Reference Standards; D015203:Reproducibility of Results; D012996:Solutions; D013379:Substrate Specificity; D013395:Sucrose / Q000032:analysis; D013997:Time Factors; D043324:beta-Fructofuranosidase / Q000378:metabolism*
chemical_list: D012996:Solutions; D005632:Fructose; D013395:Sucrose; D043324:beta-Fructofuranosidase; D005947:Glucose
keywords: Carbohydrates; Invertase; Michaelis-Menten; Thin-layer chromatography