Depending on the pH at which such phosphatases have optimal activity, between acidic phosphatases also called acid phosphatases and alkaline phosphatases can be distinguished. Acidic phosphatases are widely distributed in many organisms, including plants.
They work optimally at approximately pH 5 without additional cofactors. In this experiment, we will extract an acidic phosphatase from bean sprout. To measure its activity, we used a substrate called disodium phenylphosphate. The more activity of acidic phosphatase has, the more phenol is produced.
So the content of phenol varies in proportion with the activity of the acidic phosphatase. The amount of phenol is determined by Folin-phenol method. However, several rearrangements of the Michaelis-Menten equation transform it into a straight-line equation.
The best known of these is the Lineweaver-Burk double-reciprocal plot. The Lineweaver-Burk plot is one way of visualizing the effect of inhibitors and determining the Michaelis Constant Km and the Maximum Velocity Vmax from a set of measurements of velocity at different substrate concentrations.
Vmax and Km are the two parameters which define the kinetic behavior of an enzyme as a function of [S]. Figure The Lineweaver-Burk double-reciprocal plot, depicting extrapolations that allow the determination of the x- and y-intercepts and slope. Preparation of the acidic phosphatase 1. Weigh 5g of bean sprout.
Then place it for 30min at room temperature. Pour the resulting homogenization through the mesh silk into a 2mL tube then centrifuge at rpm for 20min. Transfer the resulting supernatant which containing the acidic phosphatase into a fresh tube.
Dilution the supernatant for 40 times with HAC buffer pH 5. Determine the Km and Vmax of the acidic phosphatase 1.
Plot a standard curve of phenol content 2. Select 9 tubes and mark them as , 0 tube is used as blank tube number Additions mL 0 1 2 3 4 5 6 7 8 0. Folin-phenol reagent 0. Measure the A of the samples and the blank tube is used for the zero setting. A is plotted against phenol content. Calculate V0 and [S] the content of the phenol in each tube. Absorbance 0. The first is the amount of enzyme. The initial velocity is proportional to the amount of enzyme molecules.
The more enzyme, the greater the initial velocity will be since more product is being formed. Another factor that greatly influences enzyme activity is temperature. Proteins are usually denatured by temperatures above 50oC. Any temperature lower than that causes an increase in enzyme activity, until the freezing point is reached. Here, freezing an enzyme often denatures it and results in a loss of catalytic activity.
However, for every enzyme, there is an optimal temperature that results in the greatest Vo. Calculated by weighted regression analysis and direct linear plotting from the absorbance data of six female rats the medium app. As demonstrated by factorial analysis of variance only Vmax is influenced by the villus position. Abstract A quantitative histochemical method to determine the apparent Km and Vmax values of rat intestinal unspecific alkaline phosphatase at different sites of the villi is described.
References 1. This will be done by determining and investigating the optimum pH and temperature at which Alkaline Phosphatase function. Theory and principles Enzymes are the most efficient catalysts known. The catalyst itself does not change or is not consumed in the reactions they catalyze. Enzymatic reactions consist of substrates and products. Enzymes are very substrate-specific. This specificity of the enzyme molecule is due to the complementary shape of the active site of the protein and the substrate.
An enzyme consists of two p References Works Cited 1. Oxford Dictionaries. Available from: www. New York: Mc Graw Hill; AG Scientific. International Union of Biochemistry and Molecular Biology. View 2 excerpts, cites methods and background. Enzymatic characterization and regulation of gene expression of PhoK alkaline phosphatase in Sphingobium sp.
Enzymatic precipitation provides a novel cost-effective and eco-friendly method for remediation of heavy metals from different industrial effluents such as tannery, electroplating, dye industries, … Expand. View 4 excerpts, cites methods, results and background. Inhibition of digestive enzyme activities by pectic polysaccharides in model solutions. Abstract The presence of dietary fiber e. Application of HR mutant alkaline phosphatase for removal of heavy metals from single-ion solutions and effluents.
Enzyme-mediated bioremediation is an eco-friendly process for removing hazardous toxic heavy metals from the environment. The potential use of mutant alkaline phosphatase HR for bioprecipitation … Expand. Study of alkaline phosphatase interaction with putrescine using multi-spectroscopic and docking methods.
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