Enzyme Browning Lab Report

Our hypothesis states that the benzoquinone absorbance rate would be faster when the pH added to the cuvettes were greater than the pH of the potato tissue. At pH 7, reaction rates were the fastest, but not at pH 10 like we predicted. We predicted that pH 4 would have the slowest to no reaction rate, but it was faster than pH 10 because the potato tissue has a pH 6 and the pH 4 was closest to the pH of the potato tissue. A possible reason our hypothesis was only partially supported was because of possible cross contamination when preparing the cuvettes by not using different disposable pipets for each pH solution, or the test tubes could have been labeled wrong which caused them to be mixed up. In future experiments, we should look at the effects of pHs closer to the pH of the potato tissue because we can infer that closer pHs to the pH of potato tissue will have a faster reaction rate causing the potatoes to turn brown

The absorbance is measured using a Plate reader and a Standard curve is generated. Also, the different types of pipetting techniques are assessed in this Assay.

Catechol, in the presence of oxygen is oxidized by catechol oxidase to form benzoquinone (Harel et al., 1964). Bananas and potatoes contain catechol oxidase that acts on catechol which is initially colorless and converts it to brown (Harel et al., 1964). In this experiment, the effect of pH on the activity of catechol oxidase was conducted using buffers ranging from pH2 to pH10. Two trials were conducted due to the first trial results being altered by an external factor. The results were acquired by taking readings every 2 minutes for 20 minutes from a spectrophotometer and then recorded on to the table. The data collected in the table were then made into graphs to illustrate the influence of pH on the catechol oxidase catalyzed reaction. After analysis, the data revealed that pH did have a significant influence on the enzyme as recorded by absorbance per minute. However, the data was collected was not accurate due to external factors, thus the results are debatable and should be experimented again for validation.

In this experiment, there were 6 different test tubes, each containing a different amount of phosphate concentration. Using a balance, 1 g of phosphate in a 400 mL beaker was measured and then inserted into a 100 mL graduated cylinder. 5 mL of ammonium vanadomolybdate (AVM) was added, along with deionized water until the solution reached to 25 mL. Then the solution was poured into one of the six test tubes. Another test tube contained 2 g of phosphate (phosphate was again measured using the balance) and the step described earlier was repeated until five of the six test tubes were filled, each with the same volume of 25 mL. The sixth test tube was filled with only deionized water, with 25 mL as the volume. A spectrophotometer was then used to determine the percent transmittance for each solution. The absorbance was also calculated using the equation A = -log (T). Each given amount of phosphate was converted into moles and then applied the value of .025 L for the volume to calculate the molarity of phosphate in each solution.

With these absorbance numbers a concentration curve was constructed and the unknown solution was determined by finding the point of absorbance on the curve.

The dark, navy blue colored graph represented the absorbance curve for the S1 sample. The red colored graph represented the absorbance curve for the S2 sample. The green colored graph represented the absorbance curve for the P1 sample. The purple colored graph represented the absorbance curve for the P2 sample. The gaps between the P2 curve was due to the oversaturation that led to the inconclusive spectrophotometer readings. The blue colored graph represented the absorbance curve for the P1 low salt sample. The orange colored graph represented the absorbance curve for the P2 low salt sample. The light blue colored graph represented the absorbance curve for the P1 medium salt sample. The light pink colored graph represented the absorbance curve for the P2 medium salt sample. The light green colored graph represented the absorbance curve for the P1 high salt sample. The light purple colored graph represented the absorbance curve for the P2 high salt

The values of color absorbance are effective because color absorbance has a linear relationship with concentration values, which in turn, allows us to easily find concentration values for many solutions. Beer’s law describes this phenomenon since the absorbance is directly proportional to concentration. We observed that as the color absorbance increased, the concentration of the FeSCN2+ complex ion increased. This is because as the FeSCN2+ concentration increases, the blood-red color becomes darker due to more presence of the blood-red FeSCN2+ ion. Therefore, the color absorbance increases because there is more blue color absorbed by the darker red color. We then graphed the absorbance and concentration values and created a line of best fit. Using the line of best fit, we were able to predict the equilibrium concentrations of the FeSCN2+ solutions and find the change required to reach equilibrium. Since we already knew the initial concentration of FeSCN2+ and since we already found the equilibrium concentration of FeSCN2+, we can calculate the change in equilibrium. Using this data, we were able to calculate the equilibrium concentration of all of the species in this lab, since we already knew the change from the initial concentration to the equilibrium change. Q is less than K because there was no initial concentration of FeSCN2+, but after the system reached

Lactose is a sugar that can be put into smaller molecules, glucose and galactose. Lactose is when you are not able to digest milk and dairy meaning that the enzyme lactase that breaks down lactose is not functioning properly. ONPG was used as a substitute for lactase because even though it is colorless it helps show enzyme activity by turning yellow. This experiment measured the absorbance ONPG when exposed to lactase within an environment of different salinity’s. The enzyme, lactase, was obtained by crushing a lactaid pill and then was added into four cuvettes. ONPG and salt solution of different concentrations were added and their levels of absorption was measured by a spectrophotometer. The results showed that higher salt concentrations have a lower level of absorption. There were 4 cuvettes and within those cuvettes that solutions within them were being tested and the results showed the more salt solution added with the lactase the lower the absorbance. The less salt solution there was a higher rate of absorbance. The data supported the hypothesis that with increasing NaCl concentration there would be a decrease in enzyme activity.

The determination of the number of thiol groups by DTNB is carried out at pH> 7.5 because the extinction coefficient is strongly pH dependent at pH values more acidic than 7.5. With an altered pH the maximal extinction may be altered, meaning that the absorbency figures will be

From the results, we can conclude that 0.9mM ONPG solution has the highest absorbance value and 0.1mM ONPG solution has the least absorbance. However, 1.0mM ONPG solution, which has the highest concentration, does not have the highest

Within an acid-base titration the titration curve resembles the strengths of the corresponding acids and bases. A strong acid will correspond with a weak conjugate base, and a weak conjugate acid will correspond with a strong base. This is based on the Bronsted-Lowry model. The weak acid will donate protons to the hydroxide ion. Weak acids will have a low Ka value, the Ka value is the tendency of the acid to dissociate:

Enzymes are high molecular weight molecules and are proteins in nature. Enzymes work as catalysts in biochemical reactions in living organisms. Enzyme Catecholase is found on in plants, animals as well as fungi and is responsible for the darkening of different fruits. In most cases enzymatic activities are influenced by a number of factors, among them is temperature, PH, enzyme concentration as well as substrate concentration (Silverthorn, 2004). In this experiment enzyme catecholase was used to investigate the effects of PH and enzyme concentration on it rate of reaction. A pH buffer was used to control the PH, potato juice was used as the substrate and water was used as a solvent.

In the experiment we used Turnip, Hydrogen Peroxide, Distilled Water, and Guaiacol as my substances. On the first activity, Effect of Enzyme concentration of Reaction Rate for low enzyme concentration, we tested three concentrations of the turnip extract, and hydrogen peroxide. For the Turnip Extract I used 0.5 ml, 1.0 ml, and 2.0 ml. For hydrogen peroxide we used 0.1 ml, 0.2 ml, and 0.4 ml. We used a control to see the standard, and used a control for each enzyme concentration used. The control contains turnip extract and the color reagent, Guaiacol. We prepared my substrate tubes separately from the enzyme tubes. My substrate tube

The purpose of this experiment was to determine the pKa of the bromothymol blue (indicator) through absorption spectroscopy. Bromothymol blue being a monoprotic acid base indicator, displays different colors at different pH because of the differences in the ratio of the conjugated acid and base form. The fraction of conjugate acid and base was interpolated for the solutions through the acquired absorbance spectrum of the bromothymol blue at various pH. The rearranged form of Henderson Hasselbalch equation was graphed as a function of pH to determine the pKa of the indicator.

The ending result of this experiment confirms that as five test tubes are lined up with the varying level of absorbance, different results in the level of absorbance will appear as well, this is visible in above table. Thus, this is due to the varying amount of water in the solution. The blank sample had a 0.30 in its level of absorbance.