Discussion 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. Comparing …show more content…
the acid-disgested and undigested sugar solutions, results show a significant difference in absorbance at 420 nm. Reviewing Table 2. Every group produced data that showed that the undigested sample had more absorbance at 420nm, thus a higher concentration of glucose according to the assay’s theory of uses (for example 0.065 vs 0.012 and 0.067 vs 0.013). Therefore in the digested solution, it can be assumed that the B-D-glucose has undergone a reaction which yields it incapable of being further catalyzed by glucose oxidase. If this were not true the digested solution would undergo oxidation and produced H2O2, which will be used to produce ferricyanide after the horseradish peroxidase. Reviewing the control samples to the altered conditions, various conclusions can be made about the ability of the enzyme to catalyze glucose. Temperature was the first parameter analyzed. Subjecting the assay to an ice water bath, the result showed a decrease in absorbance verses the 38.9°C experiment. Table 6. shows that the average absorbance for the 39.8°C assay is 0.261 with a glucose concentration of 151 mM, which is significantly higher than the ice bath’s 0.1825 absorption and 108 mM concentration. Considering the solutions used in the ice bath and 38.9°C experiment were the identical, it can be assumed that the temperature has some effect on how the species present interact with the enzyme(s) of the assay. The most plausible answer for this would be that there are less interactions between substrate and the enzyme present in the colder assay, thus producing less ferriccyanide, and absorbance in the process. In contrast, at the higher temp of 40°C there are more interactions, driving the reaction(s) to produce ferriccyanide. Consequently, differences in temperatures can skew the results of the assay. In a real world setting the assay should be calibrated and used at a particular temperature in order to yield accurate and replicable results. The amount of enzyme present is shown to have an effect on the production of ferricyanide, considering there is a difference in absorbance. 0.277 vs 0.247, shown in Table 5. Conceptually the enzymes in either reaction are being recycled, and can continuously produce products as long as the correct substrates are present. Therefore in theory, if both the 0.5 mL and 0.1 mL experiments were given ample amount of time (beyond the 45 mins used in these assays), both should produce the same amount of products. This would yield identical amounts of ferricyanide, giving the same absorbance and glucose concentration. This is not the case however, considering the difference in calculated glucose concentration is 160 mM verse 144 mM. The ability for the enzyme to act upon the substrate in the key factor in comparing the different volumes of enzyme tested. More enzyme, such as in the 0.5 mL sample, is able to quickly convert substrate to produce, as opposed to the 0.1 mL that is slower to produce. Both, high or low concentration of enzymes could therefore, catalyze 1 mol of substrate, but the higher would deplete the substrate quicker. Therefore, in a real world setting a standardized amount of enzyme should be used in all assay to produce accurate results. Furthermore, a high concentration of enzyme can produce results quicker given there are more catalyst present to drive the reaction. The amount of time that the assay are allow to react with the sample also can affect product yield, thus absorbance and calculated glucose concentration within the context of this lab. There are two tables above presented for this data, Table 4.1 and Table 4.2. Noting the large differences in results from the two, they were separated to produce more accurate averages and lower standard deviations in their own respect. Table 4.2 does not seem to fit well with the other samples, considering the absorbance data for samples 8, 9, and 10 seen in Table 2. Both tables however, exhibit the effects of reaction time on ability to produce product. In Table 4.1, the glucose concentration of the 45 minute reaction is 158 mM compared to the 5 min reaction which has a concentration of 149 mM. This data shows that most of the product produced by the 45 minute reaction is produced within the first 5 mins of the reaction. Therefore, a set time should be standardized for all assays to ensure accurate and comparable data. End of lab report Questions: **Please note, these questions are intended to have you think about the theory behind the experiment (similar to how you would write your introduction) 1. What is the role of the horseradish peroxidase in today’s experiment? Why is this important? (Be specific to what the horseradish peroxidase does and what it allows you to determine.) (5 points) Horseradish peroxidase acts as an enzyme that catalyzes a reaction between ferrocyanide and hydrogen peroxide.1 The product of the reaction is water and ferricyanide, which has a yellow color.
This yellow species can then be measured using UV absorbance (max abs = 420 nm), and thus the concentration of the can species determined.1 Horseradish peroxidase in important in the glucose assay because it catalyzes a reaction that includes one of the products from the glucose oxidase reaction, H2O2. There will be one H2O2 produced for every oxidized B-D-glucose, which will then be used to oxidize one ferrocyanide into the one measurable ferricyanide. Therefore, using the enzymes glucose oxidase and horseradish peroxidase in a consecutive manner, users can determine the concentration of glucose present in solution by simply measuring the amount of ferricyanide produced because of it (this is a one to one ratio). 2. If this experiment were designed to determine the amount of Fructose in a solution, describe what, if anything, would need to change in the reaction? Explain why there would or would not need to be changes. (5 …show more content…
points) Glucose oxidase would have to be able to oxidize fructose, and produce hydrogen peroxide for the horseradish to then use. The substrates, glucose and fructose, have some similarity but the biggest difference being that glucose is a six membered ring where fructose is five. An enzyme that works on one would be unlikely to work on the other due this difference in structure considering the lock and key model. Therefore the active site of the enzyme would have a change in order to accommodate fructose, thus neglecting it’s a ability to interact with glucose. However, assuming some enzyme presented an active site that resembled a low energy transition state for both substrates, then one enzyme in theory could interact with both, thus presenting competitive competition. 3.
What is the transducer in our sensor scheme (in the assay you used)? What is the most common transducer for commercial glucometers (such as what you would buy at a drug store)? May have to research this. (5 points) The transducer in the assay was the Shimadzu UVmini-1240 UV-Vis Spectrophotometer. It is used to measure the absorbance of ferricyanide in solution. Ferricyanide is a yellow species that be measured and compared to the glucose concentration of the sample. Electrochemical glucometers look like the most common type of transducer for commercial use. It utilizes electrodes and flowing current measured by a voltmeter.2 4. Briefly describe an alternative technique that could be used to measure the amount of glucose within sports drinks. (5 points) There is a technique called polarimetry that uses polarized light and asymmetric carbons like those found in glucose. The amount of polaritization can be used to determine the amount of active molecules present in solution.3 The equation used is measured rotation of angle = (a)lc, (a) is optical activity, c is the concentration, and l is the pathlenght. The concept seems similar to Beers
law.
These labels indicated the lactose solution that was be placed into the mini-microfuge tubes. The varying lactose ph solutions were obtained. The four miniature pipets were then used, (one per solution,) to add 1mL of the solution to the corresponding mini-microfuge tubes. When this step is completed there were two mini-microfuge tubes that matched the paper towel. Then, once all of the solutions contained their respective lactose solutions, 0.5mL of the lactase enzyme suspension was added to the first mini-microfuge tube labeled LPH4 on the paper towel, and 4 on the microfuge tube. As soon as the lactase enzyme suspension was added to the mini-microfuge tube, the timer was started in stopwatch mode (increasing.) When the timer reached 7 minutes and 30 seconds, the glucose test strip was dipped into the created solution in the mini-microfuge tube for 2 seconds (keep timer going, as the timer is also needed for the glucose strip. Once the two seconds had elapsed, the test strip was immediately removed, and the excess solution was wiped gently on the side of the mini-microfuge tube. The timer was continued for 30 addition seconds. Once the timer reached 7:32 (the extra two seconds accounting for the glucose dip), the test strip was then compared the glucose test strip color chart that is found on the side of the glucose test strip
Finally, the last part of the experiment examined the enzyme activity at different pH levels. Four sets of 11 tubes were set up in this part. The procedure for this part is the same as before, but 4 other buffers were substituted for the standard pH 7.3 phosphate buffer. Set A used the 5.5 pH buffer while set B used the 6.5 pH buffer. The buffer of pH 8.5 was used for set B and for set D the pH was 9. The absorbance readings for 4 sets were taken and recorded in table 13. Using the linear equation that the best-fit line gave for each set, the Km and the Vmax of each set were determined. Then, table 15 was made by dividing the Vmax by the Km. of the four pHs. The Vmax and Km of the control set were also used to make
Figure 3: The absorbance of peroxidase reactions over two minutes using pH 3, pH 5, pH 7, and pH 9.
“Peroxidase is a heme-containing enzymes found in peroxisome (eukaryotic organelle) and can be obtained from a variety of plant tissues” (Coleman, 2015). Peroxidase breaks down different compounds and adds hydrogen to make it harmless. Peroxidase was a number of substrates such as cytochrome C and many more dyes (Ahmad, 2014). PH is 7 which is neutral and what is does to higher or lower the activity results in activity loss of an enzyme. The purpose of this lab is to observer effects different environments have on an enzymes (Lockwood, 2012). If the pH is changed then the speed of the enzymes will change (Urry, 2013). Prediction when the pH is changed of course it will speed up the enzymes because the pH changes the enzymes shape and when changing the enzymes shape is affects the function of the chemical
This lab attempted to find the rate at which Carbon dioxide is produced when five different test solutions: glycine, sucrose, galactose, water, and glucose were separately mixed with a yeast solution to produce fermentation, a process cells undergo. Fermentation is a major way by which a living cell can obtain energy. By measuring the carbon dioxide released by the test solutions, it could be determined which food source allows a living cell to obtain energy. The focus of the research was to determine which test solution would release the Carbon Dioxide by-product the quickest, by the addition of the yeast solution. The best results came from galactose, which produced .170 ml/minute of carbon dioxide. Followed by glucose, this produced .014 ml/minute; finally, sucrose which produced .012ml/minute of Carbon Dioxide. The test solutions water and glycine did not release Carbon Dioxide because they were not a food source for yeast. The results suggest that sugars are very good energy sources for a cell where amino acid, Glycine, is not.
When the Coris began to study carbohydrate metabolism, it was believed that glucose, a type of carbohydr...
Record any color changes of the strip and determine the glucose concentration according to the key on the bottle.
First is the Benedict’s test for reducing sugars which determines if a carbohydrate contains a free aldehyde or ketone group. When Benedict’s reagent is heated with a reactive sugar the color of the reagent changes. The initial solution color of the Benedict’s regent is sky blue. Depending on the number of available sites for the reaction to occur, the reaction will result in a solution that may range from green to yellow to orange to brick red, a red precipitate may form if more time is allowed. The test will only show a positive reaction for starch if the starch has been broken down to maltose of glucose. ("BIO 1510 Laboratory Manual," 2016)
In this experiment, we will explore the properties of fresh potato extract in Phosphate buffer pH6 containing the enzyme polyphenol-oxidase and measure the different concentration of this enzyme activity by observing the production of pink/gold melanin, when 0.1% catechol and phosphate buffer pH6 is mixed into the solution. At this stage of the experiment, we are assuming that all other variables that can act as inhibitors of the enzymatic activity such as temperature or pH levels are under control. Fruits and vegetables are known to have small amounts of catechol and polyphenol-oxidase (enzyme), which are the cause of the production of browning effect in the out-layer skin or liquid of the fruit or vegetable when it is damaged. Polyphenol-oxidase
Experimental Strategy: In this experiment, the yeast being used is called Saccharomyces cerevisiae. This type of yeast follows fermentation which is very unique and can tell how much carbon dioxide is produced by fermentation more accurately compared to cellular respiration. Three test tubes will be filled with a specific volume and concentration of sugar with a certain amount of yeast in each test tube. Two of the three test tubes will have similar concentrations of sugar with different amounts of yeast...
Kirk, Julienne., Stegner, Jane., 2010. Journal of Diabetes Science and Technology: Self-Monitoring of Blood Glucose: Practical Aspects. Retrieved from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2864180/
In essence, the main objective was to use chemical titration to measure and then calculate the rate of conversion of hydrogen peroxide (H2O2) to water and oxygen by using the enzyme catalase. Other purposes of the lab were; to measure the effects of changes of temperature, pH, enzymes concentration, and substrate concentration on rates of an enzyme. The lab was also an opportunity to see a catalyzed reaction in a controlled experiment. And the last objective was to learn how environmental factors affect the rate of enzyme catalyzed reactions.
Molisch test is one of the useful qualitative test for presence of carbohydrates in solution. The three glucose solutions all have a violet colored ring formed at the junction between the two layers. This showed that carbohydrates are present in these sugar solution. This test involved the addition of concentrated sulphuric acid which causes dehydration of all carbohydrates to give ‘furfural’ compound, where pentoses are dehydrated to furfural, and hexoses are dehydrated to 5-hydroxymethylfurfural (Molish's reagent, 2009). These compounds will later react with – naphtol which is Molisch reagent to give a purple colored complex. The test has to be carried out slowly as the violet colour formed at the surface of contact of concentrated sulfuric
One major change I would make into the procedure is the to make a more efficient way to extract the sugar. To make it more efficient fi...
If glucose is present in urine then when adding benedict’s solution to the urine the color will change to a greenish or red-brown color.