Paranitrophenol Phosphate Reaction
Enzymes are found in all living cells as part of their protein constitution, which means that they are made of chains of amino acids bound together. These molecules provide energy for the organisms by catalyzing various biochemical reactions. Most of these chemical reactions would not take place in the conditions available if the enzymes were not a present. Therefore, we can say that being involved as catalysts is the main and most important role of an enzyme in any organism. Furthermore, many reactions that are thermodynamically favored do not occur quickly because there is not enough energy necessary to start the reaction (1). This energy is called the activation energy and the role of the enzyme is to provide energy input to reach that threshold
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In this experiment, a reaction is considered where a colorless Paranitrophenol Phosphate (PNPP) is converted to the yellow paranitrophenol (PNP) through the action of an enzyme called alkaline phosphatase. This enzyme normally acts on molecules that have a phosphate group by cleaving it. The main purpose of the experiment is to study the kinetics of alkaline phosphatase when acting on a conversion of PNPP to PNP by measuring the activity of the enzyme in terms of the amount of the product made. Since PNP has a yellow color, we make use of the spectrophotometry technique for absorbance at 420 nm to keep track of how much product is there (6). The experiment is performed in three main segments: in the first one, a standard curve of PNP absorbance in terms of nmoles of PNP is generated so that all absorbance values obtained later on in the experiment can be converted to a concentration value. The second part of the experiment is an enzyme optimization assay so as to determine the best enzyme concentration to be used for the rest of the assays. In the third segment, once the enzyme concentration is fixed, the substrate concentration is then varied as so to obtain the Michaelis-Menten and Lineweaver-Burk data by determining the velocity in terms of substrate concentration, and eventually determining values for Vmax, Km and the specific activity as an ultimate purpose of this experiment. Vmax is the maximal velocity of the reaction that is reached when all the enzymes are being bound to the substrate and no more are available. Km is a parameter that represents the substrate concentration that corresponds to half of the maximal velocity Vmax. Km is also a good measurement of the affinity of the enzyme where the higher its value, the lower is the affinity (3, 4,
In this experiment, a reaction is considered where a colorless Paranitrophenol Phosphate (PNPP) is converted to the yellow paranitrophenol (PNP) through the action of an enzyme called alkaline phosphatase. This enzyme normally acts on molecules that have a phosphate group by cleaving it. The main purpose of the experiment is to study the kinetics of alkaline phosphatase when acting on a conversion of PNPP to PNP by measuring the activity of the enzyme in terms of the amount of the product made. Since PNP has a yellow color, we make use of the spectrophotometry technique for absorbance at 420 nm to keep track of how much product is there (6). The experiment is performed in three main segments: in the first one, a standard curve of PNP absorbance in terms of nmoles of PNP is generated so that all absorbance values obtained later on in the experiment can be converted to a concentration value. The second part of the experiment is an enzyme optimization assay so as to determine the best enzyme concentration to be used for the rest of the assays. In the third segment, once the enzyme concentration is fixed, the substrate concentration is then varied as so to obtain the Michaelis-Menten and Lineweaver-Burk data by determining the velocity in terms of substrate concentration, and eventually determining values for Vmax, Km and the specific activity as an ultimate purpose of this experiment. Vmax is the maximal velocity of the reaction that is reached when all the enzymes are being bound to the substrate and no more are available. Km is a parameter that represents the substrate concentration that corresponds to half of the maximal velocity Vmax. Km is also a good measurement of the affinity of the enzyme where the higher its value, the lower is the affinity (3, 4,