L.11 Cytochrome C Comparison Lab

PROTEINS AND EVOLUTION

Genes are made of DNA and are inherited from parent to offspring. Some DNA sequences code for mRNA which, in turn, codes for the amino acid sequence of proteins. Over time, random mutations in the DNA sequence occur. As a result, the amino acid sequence of Cytochrome C also changes. Cells without usable Cytochrome C are unlikely to survive. The cytochrome C is a small protein found loosely associated with the inner membrane of the mitochondrion. It is found in eucariotic cells and has an hemeprotein. It is essential to the electrone transport chain and it is involved in using energy in the cell (ATP).

The purpose of this practice is to compare the relatedness between organisms by examining the amino acid sequence in the protein.

METHOD

First, we compared the amino acid sequence of Cytochrome C in various organisms:
-horse, donkey, whale (mammals)
-penguin, chicken (birds)
-snake (reptile)
-moth (insect)
-wheat (plant)
-yeast (fungi)

1- We marked the amino acids which were different.
2-Then we counted and recorded the total number of differences.
3- We shared the data with the rest of the class to complete Table 1.


After that, we made a cladogram (branching tree)
1- The two most closely related species had the fewest differences in amino acid sequence. We placed the two most closely related species on the two shortest branches of the tree.
2-Then we placed the next two closest species on the next shortest branches.
3- And we continued until all the species had been placed.

RESULTS AND OBSERVATIONS

CONCLUSIONS

There are 0 differences between chickens and turkeys in their Cytochrome C amino acid sequence.

We think that the horse and the zebra have 1 or 2 differences, like the donkey and zebra.
To make this prediction we used this information: if they can reproduce and if the offspring will be fertile or not, we compared organs, compared embrios...

More closely related organisms have more similar Cytochrome C because evolutionarily, it hasn't been that long since they separated. If the species are close, it means that less time has passed since they separated (there are less accumulated mutations). They have a common ancestor.

Other data, including genes, suggests that fungi are more closely related to animals than plants. But Cytochrome C data suggests that fungi, plants and animals are equally distantly related because if there are more than 40 genes, there are too many mutations to see it clearly.

L9. Protein identification

On december 22nd we did an experiment about proteins. The objectives are to identify the peptide bonds and to compare the protein concentration in different foods.

Biuret's test
The Biuret's test is a chemical test used for detecting the presence of peptide bonds (the bonds that link together amino acids that form chains --> proteins). A peptide bond can be broken by hydrolysis. The intensity of the colour of the Biuret's test is directly proportional to the protein concentration. The solution to be tested in treated with a strong base followed by a few drop sof copper II sulphate. If the solution turns purple protein is present. Only peptides with a chain of at least 3 amino acids give a significant measurable colour shift with these reagents (polypeptides).


MATERIALS

-250 ml beakers
-Test tube rack
-6 test tubes
-6 x 10 ml pipet
-Mortar
-Glass marking pen
-Gloves
-Goggles
-Milk
-Rice milk
-Egg (white and yolk)
-Yogurt
-Potato
-Distilled water
-NaOH 20%
-10 drops of CuSO4

PROCEDURE

First of all we had to dilute the protein:
1- We added 100 ml of distilled water to each 250 mL beaker, and with the glass marking pen we labeled them as M (milk), R (rice milk), EW (egg white, EY (egg yolk), Y (yogurt) and P (potato).
2- Then we added 10 ml of a dispersion of each food in the belonging beakers, we separated the egg white and yolk in two different beakers and mashed the potato.

Then we prepared the samples:
3- We cleaned and dried the test tubes and labeled them like the beakers. Each group used the dispersions from the same beakers. Then we added 2 mL of every food dilution of the beaker to each corresponding test tube.
4- After that we added 2 mL of 20%NaOH dissolution to each test tube, Andrea did this because NaOH is caustic so she had to put gloves and goggles on.
5-We shook gently and added 5 drops of CuSO4 in each test tube. Then we allowed the mixture to stand for 5 minutes.
6-Finally we saw the colour change (to pink or purple) amd we compared the test tubes.

Results and observations:
Milk - positive
Rice milk - negative
Egg white - positive
Egg yolk - negative
Yogurt - positive (it has less water, the protein is more concentrated)
Potato - positive, but our teacher said that it had to be negative because it had starch.

We saw that all the foods of animal origin gave a positive result, they all have proteins. Rice milk is negative because it has starch like the potato (but strangely the potato was positive). Also, we ordered the food from more concentration of protein to less according to the colour: 1- egg white, 2- yogurt and potato, 3- milk.

QUESTIONS

1- Which food has proteins?
Eggwhite, milk yogurt and potato.

2- Which food has more proteins? Why?
Eggwhite because it's animal food and it's rich in proteins. Then the yogurt and potato and finally the milk.

3-Do you find any difference between rice milk and cow milk?
Rice milk doesn't have proteins however cow milk does as i had said before.

4- Is there any difference among milk and yogurt? why?
Yes, yogurt has more protein than milk because the protein is more concentrated than in the milk, it has less water.