Methods:Centrifugation:20 Grams of refrigerated cauliflower was obtained and ground up with 40mL of Mannitol Grinding Buffer and then filtered. 2mL of this solution was separated as F1 and the remaining was centrifuged (100x gravity for 30 min. at 4o C). Two distinct layers were created after centrifugation (pellet 1 or P1 and supernatant 1 or S1). P1 was placed in ice with 8.0 mL of Mannitol Assay Buffer. 2mL of S1 was stored and the rest was centrifuged (10,000 x gravity for 30 min. at 4oC). After centrifugation, S1 formed 2 layers (pellet 2 or P2 and supernatant 2 or S2). 8.0 mL of Mannitol Assay Buffer was added to P2Microscopy:One drop of each fraction (F1, S1, P1, S2, P2) was placed on the slide with 1 or 2 drops of Azure C dye and observed under the microscope. Pictures were taken of the observations.Succinate Dehydrogenase (Part 1):9 different cuvettes were obtained and each cuvette was filled with different concentrations of various solutions (Assay Buffer, Azide, DCIP, Malonate, and Succinate). 3 of the 9 were control cuvettes (Malonate, Succinate, and Azide). The Spectrophotometer was set to 600 nm wavelength and each of the cuvette’s OD/ absorbance was measured after the addition of 0.36 mL of the fraction samples. The OD was read at 0 and 3 minutes intervals of the addition of the fraction sample to each cuvette. (Part 2):10 new cuvettes were obtained and various concentrations of Mannitol Assay buffer were added to each cuvette (P2, P2A, P2B, P2C, & S2). Then 0.2 mL of DCIP and 0.2mL of Succinate were added to all the cuvettes. The Spectrophotometer was set at 600 nm wavelength and the absorbance values were observed for each of the cuvette after 0 and 3 minutes of adding the fraction samples from P2.Calculating the Velocity of DCIP Reduction:First the OD reading of 3 minutes and 0 minutes was found for each sample Δfraction (Table 3 & 4). Then the found was divided by DCIP extinction coefficient Δ(21mM-1cm-1) to find the concentration of DCIPreduced. Then the amount of the DCIPreduced was found by multiplying the concentration found above with the volume of reaction. Finally, to find the velocity, moles of DCIPμ reduced was divided by the reaction time.

Results:Table 3: OD absorbance of Sample Fractions through SpectrophotometerSample Fraction

OD Reading: 0 Mins

OD Reading: 3 mins

Concentration(mM)

Velocity ( μmoles/min)

Filtrate 0.519 1.578 1.0086 * 10-4 3.3619 * 10-5

S1 0.839 0.994 1.4762 * 10-5 4.9206 * 10-6

P1 1.767 1.685 -7.8091 * 10-6 -2.603*10-6

S2 0.776 0.585 -1.81905*10-5 -6.063*10-6

P2 0.533 0.544 1.04762*10-6 3.4921*10-7

Malonate 0.803 0.524 -2.65714*10-5 -2.65714*10-5

Succinate 0.463 0.425 -3.61905*10-6 -1.206*10-6

Azide 0.670 0.731 5.80952*10-6 1.9365*10-6

Table 3: As discussed in methods, various concentrations of Mannitol Assay Buffer, Azide, DCIP, Malonate, Succinate, and subcellular fractions were added to the cuvettes (Table 1). The cuvettes were then placed in a spectrophotometer and the OD was read at 0 minutes of adding the sample fractions and at 3 minutes.

Table 4: OD absorbance of varied P2 Sample fractions through spectrophotometerSample Fraction

OD Reading: 0 Mins

OD Reading: 3 Mins

Concentrations (mM)

Velocity ( μmoles/min)

P2 0.649 0.782 1.26667*10-5 4.2222*10-6

P2A 0.955 0.996 3.90476*10-6 1.3016*10-6

P2B 0.647 0.730 7.90476*10-6 2.6349*10-6

P2C 0.649 0.676 2.57143*10-6 8.5714*10-7

S2 0.749 0.948 1.89524*10-5 6.3175*10-6

Differing concentrations of Mannitol Assay Buffer, Azide, Malonate, Succinate, and P2 subcellular fractions were added to 5 cuvettes (Table 2). The cuvettes were placed in the spectrophotometer and the OD was read at 0 minutes and then at 3 minutes after the addition of the sample fractions.

Figure 1. Supernatant 2 (S2) Sample under a microscope

Figure 1: One drop of the sample S2 was dropped on a slide. Then 2 drops of Azure C were added to the slide. The slide was then observed under a 40x magnificationFigure 2: Filtrate (F1) sample under a microscope

Figure 2: One drop of the sample F1 was placed on a slide along with 2 drops of Azure C. The slide was then observed under the microscope at a 40x magnification

Figure 3. Pellet 1 (P1) Seen under a microscope

Figure 3: 1 drop of P1 was placed on a microscope slide with Azure C staining. The sample was then observed under a microscope was a 40x magnification.Figure 4. Supernatant 1 (S1) Under a microscope

Figure 4: 1 drop of S1 was placed on a microscope followed by 2 drops of azure C staining dye. The sample was observed under a microscope with a 40x magnification.

Filtrate:The filtrate solution had a reading of 0.519 at 0 mins and a 1.578 reading at the 3 minutes mark. The reduction velocity of DCIP was found to be 3.3619 * 10-5 μmoles/min. Under the microscope, purple staining was observed (figure 2).

S1:This specific solution had an absorption of 0.839 at 0 mins and a 0.994 reading at 3 mins. The reduction velocity of DCIP was found to be 4.9206 * 10-5 moles/min. μPurple staining was observed under the microscope (figure 4).

P1 :The Pellet 1 solution had an OD of 1.767 at 0 mins and a 1.685 reading at 3 mins. The reduction velocity of DCIP was found to be -2.603 * 10-6 moles/min. Under theμ microscope, purple staining was observed (figure 3).

S2 (Table 1):The supernatant 2, in the spectrophotometer, had the absorption of 0.776 at 0 mins and a 0.585 reading at 3 mins. The DCIP reduction calculated for S2 was -6.063 * 10-6

moles/min. Though unclear, some colored solid masses were seen under the μmicroscope (figure 1).

P2 (Table 1):This specific solution had an absorption of 0.533 at 0 mins and a 0.544 reading at 3 mins. The reduction velocity of DCIP was found to be 3.4921 * 10-7 moles/min. μ

Malonate Control:

The Malonate solution had an OD of 0.803 at 0 mins and a 0.524 reading at 3 mins. The reduction velocity of DCIP was found to be -2.657 * 10-5 moles/min.μ

Succinate Control:The Succinate Control, in the spectrophotometer, had the absorption of 0.463 at 0 mins and a 0.425 reading at 3 mins. The DCIP reduction calculated for the control was -1.206 * 10-6 moles/min.μ

Azide Control:The filtrate solution had a reading of 0.670 at 0 mins and a 0.731 reading at the 3 minutes mark. The reduction velocity of DCIP was found to be 1.9365 * 10-6 μmoles/min

P2 (table 2):This specific solution had an absorption of 0.649 at 0 mins and a 0.782 reading at 3 mins. The reduction velocity of DCIP was found to be 4.22* 10-6 moles/min (Table μ4)

P2A:The filtrate solution had a reading of 0.955 at 0 mins and a 0.996 reading at the 3 minutes mark. The reduction velocity of DCIP was found to be 1.3016 * 10-6 μmoles/min

P2B:The sample of P2, in the spectrophotometer, had the absorption of 0.647 at 0 mins and a 0.730 reading at 3 mins. The DCIP reduction calculated for P2B was 2.6349 * 10-6 moles/min.μ

P2C:The filtrate solution had an OD reading of 0.649 at 0 mins and a 0.676 reading at the 3 minutes mark. The reduction velocity of DCIP was found to be 8.5714 * 10-7 μmoles/min.

S2 (Table 2):The supernatant 2, in the spectrophotometer, had an absorption of 0.749 at 0 mins and a 0.948 reading at 3 mins. The DCIP reduction calculated for S2 was 6.3175 * 10-

6 moles/min.μ