The goal of this experiment was to determine the Michaelis constant (Km) and also the maximal velocity (Vmax) and the inhibition of alkaline phosphate. In order to accomplish these goals, 5 samples were used. Each sample contained different volumes of 0.2 m MPNPP (p-nitrophenylphosphate) and 0.2 M Tris-Hcl at a pH of 8.0. To each sample 0.2 mL of the enzyme studied (Alkaline Phosphatase) were added upon insertion on the spectrophotometer apparatus. With intervals of 20 seconds their absorbance at a wavelength of 410 nm was recorded at time frame of 2 minutes for each solution. The initial velocity of the reaction (Vo) for each of the 5 samples was determined by plotting the change on the absorbance versus time. The slope was found from the
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They catalyze most of the reactions compromised in metabolism and they do not just make the majority of the reactions possible, but also serve as mechanism of stabilization and control for these reactions. It is fair enough to admit that without the presence of enzymes life would not be possible. Enzymes kinetics focuses on how the enzymes behave in response to different concentrations of both substrates and products. During this lab the kinetics of the enzyme alkaline phosphatase will be explored. Alkaline phosphatase is considered as an ubiquitous enzyme which can be obtained by isolation from kidney, bone, plasma, intestine, liver, among others. It catalyses the removal of a phosphate. The reaction that will be based on this lab is the removal of a phosphate that comes from the substrate molecule, p-nitrophenolphosphate (PNPP). While the substrate has no color, the product p-nitrophenol is yellow, and this give the advantage of following the reaction mechanism. The reaction progress can be appreciated by measuring as the yellow color forms. Substrates that are able to change their color, in this case are called chromogenic substrates, which make the study of enzymes way easier. The absorbance will increase at a linear rate as more PNPP is formed. As a determinate reaction slows down the absorbance may eventually get to a plateau, were less product is formed. The velocity of the reaction is highly affected by the substrate concentration
They catalyze most of the reactions compromised in metabolism and they do not just make the majority of the reactions possible, but also serve as mechanism of stabilization and control for these reactions. It is fair enough to admit that without the presence of enzymes life would not be possible. Enzymes kinetics focuses on how the enzymes behave in response to different concentrations of both substrates and products. During this lab the kinetics of the enzyme alkaline phosphatase will be explored. Alkaline phosphatase is considered as an ubiquitous enzyme which can be obtained by isolation from kidney, bone, plasma, intestine, liver, among others. It catalyses the removal of a phosphate. The reaction that will be based on this lab is the removal of a phosphate that comes from the substrate molecule, p-nitrophenolphosphate (PNPP). While the substrate has no color, the product p-nitrophenol is yellow, and this give the advantage of following the reaction mechanism. The reaction progress can be appreciated by measuring as the yellow color forms. Substrates that are able to change their color, in this case are called chromogenic substrates, which make the study of enzymes way easier. The absorbance will increase at a linear rate as more PNPP is formed. As a determinate reaction slows down the absorbance may eventually get to a plateau, were less product is formed. The velocity of the reaction is highly affected by the substrate concentration