Enzyme
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Examination of the Effects of Inhibitory and Non-Inhibitory Competition, Enzyme-Substrate Concentration, Along with Varying Temperature and pH-Balanced Environments on the Enzyme-Catalyzed Reaction of pNPP
Abstract:
Introduction:
Many of the chemical reactions, which take place in in living things are controlled by enzymes. In such cases, the enzyme is a protein in the cell which lowers the activation energy of a catalyzed reaction, which serves to increase the rate of the reaction.
Alkaline phosphatase is made throughout the body. Its function is to remove phosphate groups from nucleotides and proteins, many enzymes have their activity controlled by the addition and removal of phosphate groups. The blood serum level of Alkaline Phosphatase is used as a marker for disease. Elevated levels (five-times higher than normal) are found in the blood serum of people suffering from various bone and liver diseases. Irritable bowel syndrome, germ-cell tumors and liver infections will raise serum levels of Alkaline Phosphatase to a lesser extent.
To quantify the amount of activity under different conditions.
To determine the effect of enzyme and and substrate concentration upon the rate of reaction.
TO analyze some of the factors that control the rate of an enzyme-catalyzed reaction
Alkaline phosphatase, an enzyme, increases the rate of reaction for the conversion of p-Nitro-Phenyl Phosphate (pNPP, the substrate) to p-Nitrophenol (pNP, product) and inorganic phosphate (product). The rate of this reaction is affected by inhibitory compounds, enzyme and substrate concentration, PH and temperature. Being that the subject reaction takes place in humans (as well as other living things), it is hypothesized that the reaction contained therein, take place at or near optimal conditions.
To demonstrate the effect of substrate concentration upon the rate of the reaction catalyzed by alkaline phosphatase;
B) to plot the velocity of the reaction versus the substrate concentration to produce the Michaelis-Menten curve; C) to plot the reciprocal of the velocity versus the reciprocal of the substrate concentration to generate the linear Lineweaver-Burk plot; and, D) to determine the KM, Vmax and kcat values for alkaline phosphatase from both of these plots.
Different tissues in the body make slightly different versions of the enzyme, called isozymes. Recall Experiment IV, BSA and Casein did not precipitate under the same conditions. In the same manner different conditions can be used to inactivate specific isozymes of Alkaline Phosphatase, the activity of specific isozymes can be measured yielding even more diagnostic information.
However, the purpose of our experiment is not medical diagnosis but rather to test the kinetic parameters of bovine intestinal alkaline phosphatase. To do this we will use para-nitrophenyl phosphate (PNPP) as the substrate. We are using this substrate because one of the products (para-nitrophenol) has a very high molar extinction coefficient; е405 = 18,800 M -1 cm -1. This allows us to measure very small quantities of the product easily. However, only the para-nitrophenolate ion absorbs.
O2NOPO32-O2NOH+HOPO32-
Previously we used Acid Phosphatase for this experiment. As the name suggests this enzyme is active at acidic pH values. Because para-
nitrophenol has a pKa of about 7.2 we needed to add KOH to the reaction to generate the anionic form of the product. This addition of KOH also denatured the enzyme, therefore we could only generate one time-point per tube.
By using Alkaline Phosphatase (active at high pH values) we will generate the anionic form of the product directly and be able to take several time points for each well.
pNP is a bright yellow at alkaline PH and can therefore be measured by a spectrophotometer.
Materials and Methods
I. A semi-quantitative estimate of enzyme activity was first performed to assess the enzymes effect in aiding the conversion of pNPP into pNP and Pi.
The wavelength of the Spec 20 was then set to 405 nm. Under suitably alkaline conditions, p-nitrophenol ionizes to p-nitrophenolate (pKa = 6.7). p-Nitrophenolate is a yellow compound that absorbs light at 400 nm. Thus, the rate of hydrolysis of p-NPP can be determined from the increase in absorbance of 405 nm light as a function of time (DA/min).
Six, Spec 20 tubes were then individually labeled and pipetted with the following concentrations, each of which included commercial buffer of pH 8.0. The respective tubes labeled 1-6 contained, 4.0 mL of buffer and 1.0 mL of pNPP; 4.0 mL of buffer and 0.0 mL of pNPP; 3.0 mL of buffer and 1.0 mL of pNPP; 2.0 mL of buffer, 1.0 mL of pNPP and 1.0 mL of sodium molybdate; 2.0 mL of buffer, 1.0 mL of pNPP and 1.0 mL of sodium phosphate; 2.0 mL of buffer, 1.0 mL of pNPP and 1.0 mL of phenyl phosphonate. With the exception of the first 2 tubes demonstrated above, 1.0 mL of alkaline phosphatase (i.e. enzyme) was added to the Spec 20 tubes. The time of the enzyme addition was recorded concurrently. All samples were then covered with parafilm and mixed thoroughly.
The tube containing 3.0 mL of buffer, 1.0 mL of pNPP and 1.0 mL of enzyme was then placed in a Spectrophotometer, which had been set to wavelength of 405 nm using a 5.0 mL sample of distilled water as a reference. The subject sample was left in the tube until the absorbance measured between 0.3 and 0.4. The time and absorbance were recorded at such time. The absorption of all tubes was then measured and recorded starting with tube 6 and ending with tube 1 (in backwards order). Under suitably alkaline conditions, pNP ionizes to p-Nitrophenolate, which is a yellow compound that absorbs light at approximately 405 nm. Coupled with the fact that nothing else in the reaction mixture absorbs light at this wavelength, the amount of p-Nitrophenol formed in the enzyme catalyzed reaction can be determined from the increase in absorbance of 405 nm light as a function of time.
II. An attempt to quantitatively determine enzyme activity was made by concocting varying concentrations of alkaline phosphatase in 6, individually labeled Spec 20 tubes. The respective samples labeled 1-6 were contrived by