title: Thermometric sensing of peroxide in organic media. Application to monitor the stability of RBP-retinol-HRP complex.
abstract: The stability of horseradish peroxidase (HRP) in aqueous and organic solvents is applied to develop a simple thermometric procedure to detect the binding of retinoic acid-HRP conjugate to retinol binding protein (RBP). Butanone peroxide (BP) in organic phase and hydrogen peroxide in aqueous phase is detected thermometrically on a HRP column, immobilized by cross-linking with glutaraldehyde on controlled pore glass (CPG). Acetone, acetonitrile, methanol, and 2-butanol are used for detection of BP, in the flow injection analysis (FIA) mode. A linear range between 1 and 50 mM BP is obtained in all the organic solvents with a precision of 5-7% (CV%). The magnitude and nature of the thermometric response is significantly different in each organic solvent. The stability of HRP in the organic phase is used to study the stability of a retinoic acid-HRP conjugate bound to immobilized RBP. The response of HRP (to 20 mM BP) in the retinoic acid-HRP conjugate is used as an indicator of the stability of the RBP-retinoic acid-HRP complex, after challenges with various organic/aqueous solvents. Both immobilized HRP and RBP are stable at least for 6 months. The effect of o-phenylene diamine on the thermometric response of HRP is also investigated. A scheme for the design of a thermometric retinol (vitamin A) biosensor is proposed.
authors.affiliation: Department of Pure and Applied Biochemistry, Centre for Chemistry and Chemical Engineering, Lund University, Sweden.
authors.forename: K
authors.initials: K
authors.lastname: Ramanathan
authors.forename: B R
authors.initials: BR
authors.lastname: Jönsson
authors.forename: B
authors.initials: B
authors.lastname: Danielsson
mesh_terms: D002138:Calibration; D004795:Enzyme Stability; D006735:Horseradish Peroxidase / Q000737:chemistry* / Q000302:isolation & purification; D006861:Hydrogen Peroxide / Q000032:analysis; D010545:Peroxides / Q000032:analysis*; D010655:Phenylenediamines; D012177:Retinol-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification; D012997:Solvents; D013379:Substrate Specificity; D013821:Thermometers; D014212:Tretinoin / Q000302:isolation & purification; D014801:Vitamin A / Q000737:chemistry* / Q000302:isolation & purification; D014867:Water
chemical_list: D010545:Peroxides; D010655:Phenylenediamines; D012177:Retinol-Binding Proteins; D012997:Solvents; D014867:Water; D014801:Vitamin A; D014212:Tretinoin; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase