An alternative approach of affinity chromatography with extremely significant results is dye-ligand affinity chromatography. In this type of affinity chromatography, dyes compose the group of ligands than are employed in the technique (Hage et al., 2012).
The initial motivation for scientists to investigate more about dye ligand affinity chromatography was given after the interactions that took place between Blue Dextran, a Cibaron Blue and dextran conjugate, which is used as a void marker in size-exclusion chromatography, and particular kinases. Until then, only purification of various proteins by size-exclusion chromatography with Blue Dextran, like for example, erythrocyte pyruvate kinase, phosphofructokinase, glutathione reductase, and several coagulation factors had been initiated. The final conclusion of these studies was that the main reason for protein binding was Cibacron Blue F3G-A, a major reactive dye (Denizli and Pişkin, 2001).
The first study, concerning the direct and covalent immobilization of Cibacron Blue on Sephadex G-200 and also the purification of yeast pyruvate kinase with this affinity sorbent, was conducted by Roschlau and Hess (1972). Later, there were many articles and reviews dealing with this concept, which has been used in a wide variety of applications in order to purify a wide range of proteins with a number of matrices having this blue ligand.
In recent years, dye ligands have been widely used in affinity chromatography and they have led to important results concerning protein purification (Labrou, 2003). The fact that they are inexpensive, easily available and immobilized constitutes some of their advantages (Silva, Graça, Reis, Santos, Almeida, Queiroz and Sousa, 2013).
In dye-ligand affinity s...
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...sing Procion Red H-8BN that was coupled Sepharose 6B (1.96 mol dye/g moist weight gel). According to their report only when Zn(II) ions were present there was quantitative binding between the enzyme and the dye-Sepharose. Binding in very low levels occurred when these ions did not exist. Chelating agents, like EDTA, in combination with a pH change were used for the enzyme elution from the column. Additionally, Cibacron Blue F3GA-attached poly(EGDMA-HEMA) microspheres were used for the partial purification of methylotropic hydroxypyruvate reductase from a bacterial extract that had not been processed. The main characteristics of these dye-affinity microspheres are that they present really good adsorption and also they can be utilized with good results for the process of big volumes of extracts or culture medium in a liquid phase, where the target protein is present.
Finally, the last part of the experiment examined the enzyme activity at different pH levels. Four sets of 11 tubes were set up in this part. The procedure for this part is the same as before, but 4 other buffers were substituted for the standard pH 7.3 phosphate buffer. Set A used the 5.5 pH buffer while set B used the 6.5 pH buffer. The buffer of pH 8.5 was used for set B and for set D the pH was 9. The absorbance readings for 4 sets were taken and recorded in table 13. Using the linear equation that the best-fit line gave for each set, the Km and the Vmax of each set were determined. Then, table 15 was made by dividing the Vmax by the Km. of the four pHs. The Vmax and Km of the control set were also used to make
Enzymes are proteins that increase the rate of chemical reaction by lowering their activation energy. The enzyme glucose oxidase is one of the most widely used enzyme as an analytical reagent due to its ability to identify the presence of glucose, its low cost and good stability. This report discusses the role of enzymes concentration in biological reactions and the catalytic activity of glucose oxidase on D-Glucose. The activity was studied by spectrophotometry and the results were first tabulated and then plotted. The results of this experiment indicate that the enzyme concentration has no major affect on the rate of
Separations are important techniques in chemistry that are used to separate various components of a mixture. They are carried out by mixing two immiscible liquids containing certain solutes together in a separatory funnel, allowing them to separate, then extracting the distinct layers that form. The ratio of the concentration of solute present in the upper layer to the concentration in the lower layer is called the partition coefficient. The efficiency of a separation is described by this partition coefficient. If the coefficients for the two layers are largely different, then the separation can be carried out in a single step. If they aren’t, a more complex process is necessary.1,2 Countercurrent chromatography is a technique used carry out separations in these kinds of cases. It uses a continuous liquid-liquid partitioning process to streamline the usual extraction procedure.
EDTA, the chelating agent that binds with magnesium, had a high absorbency and strong color change to red. The correct cofactor was copper which with the chelating agent of PTU and citric acid which both bind strongly to copper which keeps it from binding with the enzyme. This was determined because in the trails, both PTU and citric acid had low absorbency and were clear or roughly clear in color. The catechol in each tube, which was the control for this experiment, allowed the cofactor that would be used in this reaction to be singled out. The way each chelating agent would affect the different cofactors displayed which was not needed for the reaction and which cofactors were needed for the reaction. An inconsistency that may have affected the data would be if the calibration tube malfunctioned in balancing the spectrophotometer to zero. There also could be errors if the calibration tube wasn’t used before each tube was tested in the spectrophotometer. The relationship of the cofactor and amount of enzyme activity would be that if the cofactor is inhibited or not, the enzyme activity would be higher if the cofactor is not inhibited but lower if it was inhibited by the chelating
The independent variable for this experiment is the enzyme concentration, and the range chosen is from 1% to 5% with the measurements of 1, 2, 4, and 5%. The dependant variable to be measured is the absorbance of the absorbance of the solution within a colorimeter, Equipments: Iodine solution: used to test for present of starch - Amylase solution - 1% starch solution - 1 pipette - 3 syringes - 8 test tubes – Stop clock - Water bath at 37oc - Distilled water- colorimeter Method: = == ==
... to demonstrate that hemoglobin attaches to the VIVO2+ ion at two locations of comparable strengths, named β and γ. This study has also proven that the interaction of red blood cells cannot be ignored when the conveyance or the pharmacological properties of a V compound is taken into consideration. In general, this paper does well in supporting the information available concerning hemoglobin. This article boosts the information available, concerning the diseases, genetics and functions of hemoglobin proteins. The authors achieve this by getting down to the basic level via the examination of the crystallographic structures of hemoglobin. This research has demonstrated novel examples associated with hemoglobin, pertaining to its processes and its purpose of movement. This study has immense implications for the grasp and the management of various diseases of hemoglobin
Introduction: Within this experiment we wish to facilitate a greater understanding of the concepts of experimental design and quantifying techniques. Specifically, this lab will allow us to gain an enhanced understanding of the isolation of a protein using differential solubility, which allows us to separate and purify various proteins using high concentrations of a specific salt so that they may be studied in great detail. Last week we separated our desired protein using ammonium sulfate. Since we have already extracted the desired protein, we will begin quantifying the amount using the Bradford Protein Assay. Because it is a dye-binding assay, we will use the spectrophotometer to measure the absorbance of various dilutions of a protein: this will comprise our standard curve. We will then compare the absorbance of our extracted protein from l...
Purpose: The purpose of this lab is to explore the different factors which effect enzyme activity and the rates of reaction, such as particle size and temperature.
Commercial Uses of Enzymes The use of enzymes to modify foods has increased vastly and these fermentations are continuing to be important, however, a new type of enzyme industry has evolved which involves the use of harvesting enzymes from microorganisms. The production of enzymes from bacteria and fungi can be isolated from the growth media and cleansed and purified as necessary. Generally in industrial processes the enzyme is immobilised which allows t h enzyme to be re-used and also enable the products to be separated easily. The production of textiles, paper, leather fruit juices and biological detergents are produced from Microbial enzymes.
= Before conducting the experiment I would conduct a simple test for the protein by placing a sample of the albumen into a test tube and add biurett reagent. This contains copper (II) sulphate and sodium hydroxide.
HPLC (High Performance Liquid Chromatography) is an analytical technique which separates a complex mixture of components into its specific individual components. It is a powerful tool in analysis, as it combines high speed with extreme sensitivity compared to traditional methods of chromatography because of the use of a pump which creates a high pressure and forces the mobile phase to move with the analyte in high speed. It is been used as a principle technology in various automated analyzers used for diagnostic purpose.
This experiment demonstrated the ability of agarose gel electrophoresis to separate the mixture of dyes into their individual components by the application of a combination of dyes to the same sample well. The experiment effectively demonstrated that the dyes where different in structure, energy, and composition. Most of the dyes where negatively charged at neutral pHs and only one with positive charge. The positive charge one moved an opposite direction compared to the other dyes.
Pauly, S. (2011, February). News from ABC: changes and challenges. Analytical & Bioanalytical Chemistry. pp. 1003-1004. doi:10.1007/s00216-010-4459-0.
Introduction: Purifying proteins is an important part of biology because it can help identify the function of that protein. Once a protein’s function has been identified, it can be manipulated to see how the function would change if the protein was changed. A common way to purify a protein is through Ion Exchange Chromatography, which is where charged proteins will bind to the beads in the column to purify it from the solution (Berg JM, 2002). The purpose of this experiment is to use Ion Exchange Chromatography to purify cellulase.
is impossible to specify a single best method to carry out a given analysis in