Introduction:Eukaryotic cells are complex and contain various membrane bound organelles including the mitochondria and the nucleus. A nucleus is a dense membrane bound organelle that contains the genetic materials and the mitochondria are organelles that are responsible for respiration and energy production activity. Chromosomes are the main component of the DNA and can be used to identify the presence of Nucleus. The succinate dehydrogenase, located on the inside of the mitochondrial membrane, is a positive indicator for the presence of mitochondria.

Each of the organelles has a specific density. DNA carrying nucleus is the densest organelle followed by mitochondria. Separation of substances with differing density can be accomplished by using centrifugation. Centrifugation uses the centripetal forces to separate two heterogeneous mixtures. For more dense substances lower speeds can separate the heterogeneous mixtures but to remove less dense substances from the solution, the solution needs to be centrifuged faster for a longer period of time.

Succinate Dehydrogenase (SDH) and its coenzyme FAD (flavin adenine dinucleotiede) create a complex E-FAD. This complex oxidizes succinate to fumarate. The reduced FADH transfers the electrons to the electron transport chain. Sodium Azide blocks the electron transport system (ETS) from receiving the electrons from FADH. DCIP is an artificial electron acceptor, which takes the electrons from FADH instead of the ETS. The reduction of DCIP can be identified by observation of the color change. The reduced form of DCIP is colorless and its oxidized color is blue.

If the solutions containing nucleus and the mitochondria have been successfully separated then there will be no DCIP reduction in (P1) pellet 1 sample, representing the presence of SDH, and there should be very little to no nucleus residue observed under the microscope for supernatant 2 (S2) and pellet 2 (P2)

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 wavelengths 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 at 600nm wavelength. The reduction velocity of DCIP was found to be 3.3619 * 10-5 moles/min. Under the microscope, purple staining was observed μ(figure 2).

S1:This specific solution had an absorption of 0.839 at 0 mins and a 0.994 reading at 3 mins at 600nm wavelength. The reduction velocity of DCIP was found to be 4.9206 * 10-5 moles/min. Purple staining was observed under the microscope (figure 4).μ

P1 :The Pellet 1 solution had an OD600 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 at OD600. The DCIP reduction calculated for S2 was -6.063 * 10-6 moles/min. Though unclear, some colored solid masses were seen μunder the microscope (figure 1).

P2 (Table 1):This specific solution, at 600nm wavelength, 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 OD600 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 at 600nm wavelength. The DCIP reduction calculated for the control was -1.206 * 10-6 moles/min.μ

Azide Control:The filtrate solution had an OD600 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, at 600nm wavelength, 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 4)μ

P2A:The filtrate solution had an OD600 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 with wavelength 600, 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 OD600 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, at 600nm wavelength. The DCIP reduction calculated for S2 was 6.3175 * 10-6 moles/min.μ

Discussion:Under the microscope, purple staining was observed for the F1 Solution. This Azure C purple staining is indicative of DNA presence meaning that pieces of nucleus were present. The OD600 values for F1 at zero minutes were 0.519 and 1.578 at 3 minutes mark. This high OD600 shows that mitochondrial succinate dehydrogenase was present.

S1 with the Azure C dye had a purple staining under the microscope. This means that the supernatant 1 had traces of nucleus matter. The OD600 at 0 mins was 0.839 and at 3 mins 0.994. The concentration was less than F1, meaning that the concentration of succinate dehydrogenase in this sample was low. This result suggests that mitochondrial traces were present.

P1 had very few scattered pieces of purple staining in the solution. This staining was observed under the microscope and suggests that there was a very low concentration of DNA i.e. nucleus present. The OD600 at 0 mins was 1.767 and 1.685 at 3 mins. This observation suggests that there was a very high concentration of succinate dehydrogenase. Most of the P1 solution showed evidence of high mitochondrial concentrations.

S2 had a 0.776 concentration at 0 mins and 0.585 OD600 at 3 mins. These values suggest very low traces of mitochondrial substances. The microscope created a green picture with what appears to be a large stained area. This might suggest that nucleus material was present.

P2 had an OD600 reading of 0.533 at 0 mins and 0.544 at 3 mins. This observation suggests that there was a very low if any concentration of succinate dehydrogenase i.e. mitochondrial material.

Different concentrations of P2 were placed in a series of solutions (P2A, P2B, P2C, & S2). For P2A the OD600 reading at 0 mins was 0.955 and at 0.996 at 3 mins. It has a velocity of 1.267 * 10-5. This velocity refers to the rate at which DCIP is reduced. P2B had the starting OD600 of 0.647 and 0.730 ending OD600. The velocity was calculated at 7.90*10-6. P2C had the starting concentration at 0.649 at 0 mins and 0.676 at 3 mins, with a velocity of 2.57*10-6. S2 had the OD600 reading of 0.749 at the start and the ending OD600 reading was 0.948 with a velocity of 1.89*10-5. The OD600 reading for all the values above unexpectedly increased from 0 mins to 3 mins. This error could be because of calibration error of the spectrophotometer. Another potential reason could be very small and limited amount of time provided for reduction of DCIP. S2 provided the best results for reduction of DCIP based on the data, as its velocity was the highest.

The results support the hypothesis. The velocity for the P1 solution was zero meaning that there was no reduction of the DCIP. This observation supports the claim that the mitochondria and the nucleus were separated successfully because there was no mitochondrial activity in the pellet 1. The S2 observations under the microscope were inconclusive since the green tint of the image obstructed the dye coloring, so presence or absence of nucleus in the P2 and S2 is unknown.