Kean Li

The interaction of Bromophenol Blue with proteins in acidic solution.

The interactions of Bromophenol Blue (BPB) with bovine serum albumin and gamma-globulin in acidic solutions were investigated by a spectrophotometric method. It was considered that the electrostatic force is the main binding force, and that the color change during the combination is due to the transformation of dye species of free acidic form into bound basic form as well as to the bathochromic and hyperchromic effects of conjugation. The formation of an isosbestic point in the absorption spectra was explained based on a new consideration about the solution equilibria. Two conditional constants, apparent binding constant and maximum binding number, were defined to express the binding ability of a dye to a certain protein under a given set of conditions, and two linear regression equations were derived to determine these two parameters and the molar absorptivity of bound dye. The Scatchard model is not appropriate in the treatment of data obtained here. The factors which influence the sensitivity of a dye binding protein assay were discussed, and the Sandell index was used to express the sensitivity of protein detection. It was found that sodium chloride concentration and acidity of the solutions have significant effect on the sensitivity of BPB protein assay.

Partition of horseradish peroxidase with maintained activity in aqueous biphasic system based on ionic liquid.

Enzyme activity and partition behavior in aqueous biphasic systems (ABSs) consisting of ionic liquid (IL) and salt (IL-ABSs) were investigated to increase our understanding of IL-ABSs and shed light on their application potential as enzyme extraction system. With horseradish peroxidase (HRP) as the model enzyme, several effects of alkylimidazolium chloride-K(2)HPO(4) ABSs on activity and partition behavior of enzyme were studied including alkyl chain length of ILs and concentrations of each component. High lyotropic ILs (1-butyl-3-methylimidazolium and 1-ethyl-3-methylimidazolium chloride) and adequate water content (>40%) were both essential for the activity maintenance of HRP in IL-ABS. 1-Butyl-3-methylimidazolium chloride ([C(4)mim]Cl) was found to be an appropriate IL for phase forming and HRP activity retaining. After optimization of phase condition, about 80% HRP amount was distributed in the IL-rich upper phase, and greater than 90% enzyme activity was obtained. Moreover, compared with the commonly used polymer-based ABSs, this [C(4)mim]Cl-ABS has a much lower viscosity, which is very beneficial to the experimental operation. Therefore, the tested IL-ABS could be considered as a potential enzyme extraction system.

Molecularly imprinted silica prepared with immiscible ionic liquid as solvent and porogen for selective recognition of testosterone

1-Butyl-3-methylimidazolium tetrafluoroborate ([Bmim]BF(4)), an ionic liquid (IL) immiscible with water, was used as a new type of solvent and porogen for the preparation of molecularly imprinted silica. The new imprinted silica was prepared by a sacrificial spacer molecular imprinting approach with testosterone as template molecule. The new covalent monomer-template complex used in the imprinting procedure was synthesized via the reaction of 3-(triethoxysilyl)propyl isocyanate with testosterone. The imprinted silica was characterized by FT-IR spectroscopy, N(2) gas adsorption-desorption isotherm and the high-resolution transmission electron microscopy. Moreover, the selective adsorption ability of the imprinted particles towards testosterone was investigated by the steady-state binding experiment with testosterone propionate as its structural analogue. Results showed that the imprinted silica obtained in this study had relatively homogenous structure with numerous mesopores, indicating that the IL used here is an excellent solvent and satisfactory porogen for the preparation of imprinted materials. Moreover, ILs are more environmentally friendly than traditional organic solvents due to their negligible vapor pressure. The imprinted silica possesses highly specific recognition property and high binding capacity towards testosterone, showing that the new imprinting technique is relatively successful.

Determination for micro amounts of nucleic acids by a resonance light scattering technique with dequalinium chloride

Based on the strong enhancement effect of nucleic acids on resonance light scattering of dequalinium chloride, the determination method for micro amounts of nucleic acids has been developed. Under the experimental conditions (5.0x10(-5) mol l(-1) dequalinium, pH 7.0, at room temperature) the linear range of this assay is 0.04-10.0 mug ml(-1) for calf thymus DNA and fish sperm DNA, and 0.04-35.0 mug ml(-1) for yeast RNA. The detection limits (3sigma) are 6.2 ng ml(-1) for calf thymus DNA, 7.4 ng ml(-1) for fish sperm DNA, and 7.0 ng ml(-1) for yeast RNA, respectively. Almost no interference can be observed from ionic strength, proteins, nucleoside, and most of the metal ions. Six synthetic samples were determined satisfactorily.

A linear regression method for the study of the Coomassie brilliant blue protein assay

The interactions of Coomassie brilliant blue G-250 (CBB) with bovine serum albumin (BSA) and gamma-globulin at low pH are investigated by a spectrophotometric method. It is considered that the binding of CBB to protein is because of the weak interactions (ionic, van der Waals, hydrogen bonding, and hydrophobic). The solution equilibria involving the binding of three dye species (blue, green, and red) to protein are treated in the same way as Ringbom model used in the treatment of complexation in analytical chemistry. Based on this treatment, the formation of an isosbestic point in the absorption spectra of CBB-BSA mixtures is discussed, two mathematical models for the description of the CBB protein assay are developed. The first model is a nonlinear equation which is rigorous in theory but unreliable in use because of its optimization procedure. The second model based on an approximation is a linear equation, it allows to estimate apparent binding constant, maximum binding number, and molar absorptivity of bound dye from assay data by a linear regression method. The results of the linear regression operations are reasonable and in agreement with experimental findings. Factors which influence the sensitivity of the CBB protein assay are studied using this method. Ionic strength and acidity are found to have significant effect on the binding of CBB to protein.

A method to determine quercetin by enhanced luminol electrogenerated chemiluminescence (ECL) and quercetin autoxidation

Quercetin greatly enhanced luminol electrochemiluminescence of quercetin in alkaline solution. When the concentration of luminol was 0.1 mol L(-1), the detection limit for quercetin was 2.0x10(-8) mol L(-1) with a linear range from 1.0x10(-7) to 2x10(-5) mol L(-1). The pH and buffer substantially affected ECL intensity. Quercetin was autoxidized in alkaline aqueous solution. The rate of autoxidation of quercetin in various pH buffers and borate concentrations were measured. Borate was found to inhibit quercetin autoxidation and compromise quercetin enhancement effect on luminol ECL to some extent. Two final autoxidation products were identified with LC-MS methods. Autoxidation process was associated with enhancement of ECL intensity. The ROS generated during quercetin autoxidation enhanced the ECL intensity.

Resurveying the Tris buffer solution: the specific interaction between tris(hydroxymethyl)aminomethane and lysozyme.

An unusual phenomenon, the specific interaction between tris(hydroxymethyl)aminomethane (Tris) and lysozyme (LZM), was demonstrated for the first time by rapid screen analysis of interactions using a quartz crystal microbalance (QCM) biosensor. This phenomenon was also observed in a surface plasmon resonance (SPR) system. Further study using high-performance affinity chromatography (HPAC) confirmed this specific interaction between LZM and immobilized Tris with an apparent dissociation constant (K(D)) of 6.7 x 10(-5)M. Molecular docking was carried out to identify possible modes of binding between LZM and Tris linked to a binding arm. The estimated binding free energy was -6.34 kcal mol(-1), corresponding to a K(D) of 2.3 x 10(-5)M, which correlated well with the experimental value. Based on the docking model, the three hydroxyl groups of Tris form intermolecular H bonds with Asp52, Glu35, and Ala107 in LZM. This study reinforces the importance of buffer selection in quantitative biochemical investigations. For a lysozyme ligand binding study, it is better to avoid using Tris when the ligands under study are weak binders.

A novel protein assay method using tetraphenylporphin tetrasulfonate (TPPS4)

Abstract A sensitive protein assay method which involves the reaction of TPPS4 with protein is described. When protein is added to TPPS4 solution, an absorption band with the maximum at 488 nm appears and the absorbance is proportional to the concentration of protein. Just Like the Soret absorption of the porphyrin, the new band is very narrow and there is no overlap at all between them, which means the free dyes would not give any background for the detection of the protein-TPPS4 complexes. A new spectrophotometric method for determination of protein has been constructed and applied to the determination of human plasma protein and urinary protein; The assay using microtiter plates has also been studied.

Separation study of cadmium through an emulsion liquid membrane.

A study of the transport of Cd(2+) ions through a tri-ndashoctylamine(TOA)-sorbital monooleate (Span 80)-oxylene liquid membrane has been performed with varying concentrations of HCl, KI, TOA, Span 80 and NaOH in the feed, membrane and stripping solutions. Maximum transport was observed at 0.01 M KI, 0.025 M HCl, 0.015 M TOA, 3% (w/v) Span 80 and 0.025 M NaOH. With this system, cadmium could be completely separated from Zn(2+), Fe(2+), Co(2+), Ni(2+), Cr(3+) and Mn(2+). The transport mechanism of this metal ions through the membrane has been discussed.

Molecularly Imprinted Polymer Film Grafted from Porous Silica for Selective Recognition of Testosterone

Abstract A type of testosterone‐imprinted polymer film grafted from porous silica was prepared by covalently binding azo‐initiators and then photo‐grafting. Elemental analysis and infrared (IR) spectroscopy attested the polymer formation. The material could be polymerized within 10–60 min with reproducible grafting kinetics, controllable film thickness, and obviously specific recognition ability to testosterone with the imprinting factor of 1.52. Due to its uniform size, spherical shape, controllable film thickness, and accessible sites near or at the surface, this polymer could serve as a sensing element, solid‐phase extraction material, or chromatographic stationary phase to selectively recognize or separate testosterone.