Enzyme Browning Lab Report

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 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

Table1: pH of the pure NaOH solution, the NaOH mixed with Benzophenone solution. The lowest data point was founded, and the average of the potential readings for all the values prior to the lowest point was calculated, taken into account getting rid of the data points that are far from the points in general. Furthermore, using the average I_0, the absorbance was calculated by the following equation.

Running the 4 diluted samples through the Spec 20 and finding the %T, between 18% and 85%, allows you to determine the absorbance as well. Concentration and absorbance for PO4-3 is now able to be compared which is shown in Figure 3. As concentration of the phosphate ion goes up, the percent absorbance goes up, this is a direct relationship. This coincides with Beer’s Law, A=abC, A being the absorbance, C being the concentration, a being the absorptivity and b being the cell path length.3

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

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.

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.

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

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.

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:

The purpose of this experiment was to test acid-base solutions to determine the absorbance intake they had do to their different pH levels. To do this, four similar samples were recorded with a different amount of NaOH added to each one, which would change the pH level from acidic to basic. Acidic solutions take in less absorbance, while basic solutions will have the most absorbance, as well as higher Ka and pKa values. The acidic solutions would have negative absorbance from 100% or higher transmittance, while the basic solutions would have low transmittance %, but very high absorbance levels. Introduction Acids-base indicators are organic weak acids or bases that change colors when they are neutralized.

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.

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.

Tables 2,3,4,5 and 6 show that as duration increased the absorbance also increased for each pH. The solution in the conical flask became darker (yellow) in time this is because the substrate, p-nitrophenyl phosphate was catalysed by acid phosphatase, releasing Nitrophenolate anion. It was the Nitrophenolate anion giving off the yellow colour; the presence of this feature increases the absorbance rate. The addition of sodium hydroxide distorted the shape of the enzyme making it no longer effective in its function.