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Biochemistry action of enzyme
Investigating the effect of enzyme concentration on enzyme activity
Investigating the effect of enzyme concentration on enzyme activity
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The purpose of this experiment was to analyze the activity and kinetic properties of an enzyme derived from wheat germ. For the first part of the experiment, we had to prepare a standard curve for the reaction product by measuring the velocity of the reaction catalyzed by acid phosphate that is extracted from wheat germ. Different enzyme concentrations were tested to see how fast they reacted. In order to quantify the amount of nitrophenol generated, six standards were prepared containing known concentrations of the product nitrophenol. In each cuvette, 1 mL of each of the standards was placed and labeled S1-S6. A spectrophotometer was then turned on and set to a wavelength of 410 nm. The absorbance of each tube was read and recorded on the Nitrophenol standard curve for acid phosphate assays …show more content…
The nitrophenol standard curve was then constructed by the plotting of A410 of tubes S1-S6 vs. the concentration of nitrophenol. The preparation of acid phosphate from wheat germ extract was previously performed by the instructor at the beginning of the class. In the second part of the lab the velocity of commercially available purified acid phosphatase was measured. Firstly, 12 test tubes were spitted in groups of six and labeled A1-A6 and B1-B6. Then 0.5 mL of 1.5% (w/v) of KOH was placed in each of the 12 tubes. Following that 5 ml of Phosphate Substrate solution were added to tube A and tube B. While tube A contained the reaction catalyzed by purified acid phosphatase, tube B contained the reaction catalyzed by the extracted acid phosphatase. From tube A 0.5 ml of the substrate solution was transferred to tube A1, likewise 0.5 mL of substrate solution was transferred from tube B to tube B1, at 0 time values each. With the use of a micropipette 50 µL of
Data from Table 1. confirms the theory that as the concentration of glucose increases so will the absorbance of the solution when examined with the glucose oxidase/horseradish peroxidase assay. Glucose within the context of this assay is determined by the amount of ferricyanide, determined by absornace, which is produced in a one to one ratio.1 Furthermore when examining the glucose standards, a linear calibration curve was able to be produced (shown as Figure 1). Noted the R2 value of the y = 1.808x - 0.0125 trend line is 0.9958, which is statistically considered linear. From this calibration curve the absorbance values of unknowns samples can be compared, and the correlated glucose concentration can then be approximated.
20.0cm3 of 0.10M ethanoic acid was pipetted into a conical flask. 3. 0.10M sodium hydroxide solution was titrated using phenolphthalein as indicator, until the solution was just turned pink. 4. A further 20.0cm3 of the same ethanoic acid solution was added to the flask and was mixed thoroughly.
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: = == ==
The Effect of Temperature on the Activity of the Enzyme Catalase Introduction: The catalase is added to hydrogen peroxide (H²0²), a vigorous reaction occurs and oxygen gas is evolved. This experiment investigates the effect of temperature on the rate at which the enzyme works by measuring the amount of oxygen evolved over a period of time. The experiment was carried out varying the temperature and recording the results. It was then repeated but we removed the catalase (potato) and added Lead Nitrate in its place, we again tested this experiment at two different temperatures and recorded the results. Once all the experiments were calculated, comparisons against two other groups were recorded.
Alkaline Phosphatase (APase) is an important enzyme in pre-diagnostic treatments making it an intensely studied enzyme. In order to fully understand the biochemical properties of enzymes, a kinetic explanation is essential. The kinetic assessment allows for a mechanism on how the enzyme functions. The experiment performed outlines the kinetic assessment for the purification of APase, which was purified in latter experiments through the lysis of E.coli’s bacterial cell wall. This kinetic experiment exploits the catalytic process of APase; APase catalyzes a hydrolysis reaction to produce an inorganic phosphate and alcohol via an intermediate complex.1 Using the Michaelis-Menton model for kinetic characteristics, the kinetic values of APase were found by evaluating the enzymatic rate using a paranitrophenyl phosphate (PNPP) substrate. This model uses an equation to describe enzymatic rates, by relating the
The last experiment was conducted to determine the effect of PH on the enzyme activity. The experiment corresponded with the hypothesis that at PH 7 the reaction would be faster. The reaction rate of PH 3 was extremely slow because the PH range is not close to the optimum PH of 7 and PH 10 was very high that it denatured the enzymes. That proves that extreme high and low PH levels would induce a low enzyme activity because the hydrogen bonds have been destroyed. (see table 4.0)
In my hypothesis I previously believed that the lower the lower the pH level the more seeds would germinate, due to the extra hydroxide. But more seeds actually germinated in the pHs closer to neutral. Some problems in the experiment was the towel drying out and the seeds no longer absorbing the pH, rewetting the towels with the solution could prevent this. Some experimenter errors that occurred is the seeds falling out of the towel which resulted in seeds germinating or not germinating. Also, air getting into the bags and drying out the towel faster could have been prevented by making sure the bag was closed. Lastly, the seeds did not get put in the bag exactly like the previous time which could have affected the germination. To improve this lab, measuring out the same amount of solution for each pH could have made it more accurate. To further this experiment we could have had another set of seeds in the same pH solutions but in the sun to see
This particular graph shows us that substrate concentration plays a role in enzyme activity as we already know and that the absorbance continued to increases after 15 minutes. With this particular data we were able to construct a graph of Substrate concentration versus Enzyme activity (slope) and we compared our graph with the data of a control group that did not added any NaCl in the solutions. This graph (figure 2) is one of the key to answer our hypothesis. In fact, with this particular graph we can definitely see that Sodium Chloride is an inhibitor of the enzyme PPO. The curve of the experimental data (green) is clearly below the control group curve, which means that the enzymatic activity slows down in presence of NaCl.
This process was repeated with all ten pH buffers and data was recorded. After the 60 time length for each process, oxygen production/accumulation was recorded by measuring the graduated cylinder that was submerged in water. One can observe that there were differences in which the rate of oxygen was produced ml/min between the group procedure and class procedure. It was noted that all pH levels that were tested between the group and class were the same as well as the enzyme catalase. Each reaction tube contained 2ml of the three part solutions to create the reaction.
All in all, this was a very fun and hands on experiment. The abiotic features, such as, the water and the soil, had drastic changes to them as well, and the biotic features; such as, the wheat grass and the anacharis plant, were majorly affected in this experiment, our hypothesis was that we thought that we could neutralize laundry detergent, vinegar and distilled water, so that there won’t be any difference in the growth of the wheatgrass, and the health of the anacharis plant between the control bottle and the two test bottles. We were also observing if one type of vinegar/laundry detergent solution polluted the water more that the other. We made the prediction that the white vinegar would pollute that water more because apple cider is made from more natural substances. My group and I were incorrect, about our hypothesis, but the prediction about the pollution was correct. Both of the wheat grass plants from both of the test columns eventually died.
For the lab experiment for testing the stability of beet cell membranes using pH, many materials were used as follows. Obtaining a beet we punch out cores, using a cork borer. After washing the cores we put each one inside a separate test tube, and added a different pH solution in each one. After 3 minutes in these exposure solutions, we took the beet out with a dissecting needle. Then transferred each beet to a separate test tube containing deionized water. After 20 minutes in these diffusion solutions, we took the beets out with a dissecting needle and discard it. We then stirred each solution in the test tube with a stirring rod, and transferred it to a cuvette. A spectrophotometer was then calibrated, and used to measure the absorbance of each exposure solution, and diffusion solution.
By comparing the absorption spectrum of phenol red in basic and acidic solutions, the red (basic) form had a much narrower and higher λ max (560nm) than the yellow form (420nm). As a result, the red form of phenol red was more sensitive with pH changes and it was used in for an assay in part B. In part B, the standard calibration curve was generated in order to determine the concentration of phenol red in the unknown 2B. From the slope, the average concentration of the unknown 2B was 0.001009 M. More importantly, the % difference of unknown trials was low (0.40%), indicating the high precision and reliability of the result. In part C, the pKa of the phenol red can be determined by plotting pH as a function of log ([A-])/([HA]). The pKa and
Njoku V, Chikezie P, Kaoje A (2013) Kinetic studies of alkaline phosphatase extracted from rabbit ( Lepus townsendii ) liver. African J Biotechnol 10:3157–3162. doi: 10.4314/ajb.v10i16.
The seed constituents i.e., proteins, starch, non- starch carbohydrates, lipids and other small molecules determines the processing quality of wheat. The protein content and types determine the end product quality like bread, biscuit, cake, chapatti and noodles etc. Wheat grains can generally be classified as having either a soft or hard endosperm texture. Soft grains are usually used for making biscuits whereas hard grains are used for pan-breads and pasta (Moss, 1973).
Fermentation Lab CEJ The scientists conducted an experiment to investigate an alternative sugar source that could produce the most CO2. They were hired by a baker, Jim Baker, to help him create a new type of bread that is both fluffy and unique using a different type of sugar rather than the traditional sugar. Making bread includes a process called alcoholic fermentation, which is a process that converts glucose, a sugar, into ethanol and CO2 (equation: C6H12O6 → 2 C2H5OH + 2 CO2 ). Alcoholic fermentation is one of the two ways the cell can produce ATP without oxygen in anaerobic cellular respiration.