muramidase and bicinchoninic-acid

The purpose of this study is to evaluate the potential of simple high performance liquid chromatography (HPLC) setup for quantification of adsorbed proteins on various type of plane substrates with limited area (<3 cm(2)). Protein quantification was investigated with a liquid chromatography chain equipped with a size exclusion column or a reversed-phase column. By evaluating the validation of the method according to guidelines of the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH), all the results obtained by HPLC were reliable. By simple adsorption test at the contact of hydrophilic (glass) and hydrophobic (polydimethylsiloxane: PDMS) surfaces, kinetics of adsorption were determined and amounts of adsorbed bovine serum albumin, myoglobin and lysozyme were obtained: as expected for each protein, the amount adsorbed at the plateau on glass (between 0.15 μg/cm(2) and 0.4 μg/cm(2)) is lower than for hydrophobic PDMS surfaces (between 0.45 μg/cm(2) and 0.8 μg/cm(2)). These results were consistent with bicinchoninic acid protein determination. According to ICH guidelines, both Reversed Phase and Size Exclusion HPLC can be validated for quantification of adsorbed protein. However, we consider the size exclusion approach more interesting in this field because additional informations can be obtained for aggregative proteins. Indeed, monomer, dimer and oligomer of bovine serum albumin (BSA) were observed in the chromatogram. On increasing the temperature, we found a decrease of peak intensity of bovine serum albumin as well as the fraction of dimer and oligomer after contact with PDMS and glass surface. As the surface can act as a denaturation parameter, these informations can have a huge impact on the elucidation of the interfacial behavior of protein and in particular for aggregation processes in pharmaceutical applications.

Topics: Adsorption; Animals; Cattle; Chickens; Chromatography, High Pressure Liquid; Chromatography, Reverse-Phase; Data Accuracy; Dimethylpolysiloxanes; Glass; Horses; Hydrophobic and Hydrophilic Interactions; Kinetics; Limit of Detection; Muramidase; Myoglobin; Protein Structure, Quaternary; Quinolines; Serum Albumin, Bovine; Surface Properties

Biofouling is of great concern in numerous applications ranging from ophthalmological implants to catheters, and from bioseparation to biosensors. In this report, a general and facile strategy to combat surface fouling is developed by grafting of amino acids onto polymer substrates to form zwitterionic structure through amino groups induced epoxy ring opening click reaction. First of all, a library of poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) hydrogels with zwitterionic surfaces were prepared, resulting in the formation of pairs of carboxyl anions and protonated secondary amino cations. The analysis of attenuated total reflectance Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy confirmed the successful immobilization of amino acids on the hydrogel surfaces. After that, the contact angle and equilibrium water content of the modified hydrogels showed that the hydrogels exhibited improved hydrophilicity compared with the parent hydrogel. Furthermore, the protein deposition was evaluated by bicinchoninic acid assay using bovine serum albumin (BSA) and lysozyme as models. The results indicated that the performance of the hydrogels was determined by the nature of incorporated amino acid: the hydrogels incorporated with neutral amino acids had nonspecific antiadsorption capability to both BSA and lysozyme; the hydrogels incorporated with charged amino acids showed antiadsorption behaviors against protein with same charge and enhanced adsorption to the protein with opposite charge; the optimal antiadsorption performance was observed on the hydrogels incorporated with polar amino acids with a hydroxyl residual. The improvement of antiprotein fouling of the neutral amino acids grafted hydrogels can be ascribed to the formation of zwitterionic surfaces. Finally, a couple of soft contact lenses grafted with amino acids were fabricated having improved antifouling property and hydrophilicity. The result demonstrated the success of amino acids based zwitterionic antifouling strategy in ophthalmology. This strategy is also applicable to substrates including filtration membranes, microspheres and nanofibers as well. It is a versatile method for amino acids grafting onto polymer substrates to construct zwitterionic surfaces and achieve antifouling properties.

Topics: Amino Acids; Animals; Biofouling; Cattle; Click Chemistry; Hydrogels; Hydrophobic and Hydrophilic Interactions; Muramidase; Photoelectron Spectroscopy; Polyhydroxyethyl Methacrylate; Quinolines; Serum Albumin, Bovine; Spectroscopy, Fourier Transform Infrared; Surface Properties

The adsorption of lysozyme and human serum albumin (HSA) onto hydrogel contact lenses was investigated as a function of lens surface charge. Anionic, cationic and non-ionic contact lenses were deposited using single protein solutions of identical pH and osmolarity. Protein deposition was analyzed using matrix assisted laser desorption ionization mass spectrometry (MALDI-ToF MS) and compared to a direct UV protein analysis method, the bicinchoninic acid (BCA) assay. The results showed remarkable consistency between the two techniques. By inference of results from analyses of sample solutions, lysozyme, a positively charged protein at physiological pH, was only detected on the anionic surface charged contact lenses, presumably a result of electrostatic interactions. Neither the cationic nor the non-ionic lenses deposited lysozyme, possibly due to charge repulsion. HSA, a negatively charged protein at physiological pH, was detected on the cationic lenses, again as a result of electrostatic interactions. The fact that HSA was not observed on either the anionic or non-ionic charged species further demonstrates the effect of charge repulsion.

Topics: Adsorption; Biocompatible Materials; Contact Lenses, Hydrophilic; Humans; Hydrogels; Hydrogen-Ion Concentration; In Vitro Techniques; Materials Testing; Muramidase; Proteins; Quinolines; Serum Albumin; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Static Electricity; Surface Properties

A quick, simple, and efficient extraction technique was developed for the removal of protein from soft hydrophilic contact lenses.. An extraction solvent consisting of a 50:50 mix of 0.2% trifluoroacetic acid and acetonitrile was used to remove protein from in vitro laboratory-deposited and human-worn contact lenses. The protein removed was analyzed using HPLC, bicinchoninic acid (BCA) analysis, and SDS-PAGE gel electreophoresis.. Extraction efficiency for lysozyme from laboratory-deposited Group IV lenses was determined to be approximately 100%. Group IV human-worn contact lenses were extracted and analyzed for lysozyme by HPLC and total protein by bicinchoninic acid (BCA) analysis. Groups I, II, III, and IV contact lenses deposited with an artificial tear protein solution and human-worn lenses were extracted and analyzed by SDS-PAGE gel electreophoresis and micro-BCA.. The ACN/TFA procedure offers a simple, quick, and efficient extraction technique for removal of protein from contact lenses for subsequent analysis.

Topics: Acetonitriles; Chromatography, High Pressure Liquid; Contact Lens Solutions; Contact Lenses, Hydrophilic; Drug Combinations; Eye Proteins; Humans; Methods; Muramidase; Quinolines; Solvents; Surface Properties; Tears; Trifluoroacetic Acid