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    Bio 120 Ex 3

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    piiish
    In the experiment, mungbean seeds were germinated in the dark to reduce photosynthesis and lessen starch accumulation. Differential centrifugation was used to separate subcellular components of the mungbean crude extract based on size and density. Chemical tests showed that particles separated into pellet I (PI) contained mostly starch and nucleic acids, pellet II (PII) contained lipids and nucleic acids, and supernatant II (SII) contained oxidative particles, nucleic acids, and proteins. The results mostly aligned with expectations, though there was some cross-contamination between fractions. Differential centrifugation produces a rough fractionation that can be further purified.

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    Bio 120 Ex 3

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    piiish
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    In the experiment, mungbean seeds were germinated in the dark to reduce photosynthesis and lessen starch accumulation. Differential centrifugation was used to separate subcellular components of the mungbean crude extract based on size and density. Chemical tests showed that particles separated into pellet I (PI) contained mostly starch and nucleic acids, pellet II (PII) contained lipids and nucleic acids, and supernatant II (SII) contained oxidative particles, nucleic acids, and proteins. The results mostly aligned with expectations, though there was some cross-contamination between fractions. Differential centrifugation produces a rough fractionation that can be further purified.

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    In the experiment, mungbean seeds were germinated in the dark to reduce photosynthesis and lessen starch accumulation. Differential centrifugation was used to separate subcellular components of the mungbean crude extract based on size and density. Chemical tests showed that particles separated into pellet I (PI) contained mostly starch and nucleic acids, pellet II (PII) contained lipids and nucleic acids, and supernatant II (SII) contained oxidative particles, nucleic acids, and proteins. The results mostly aligned with expectations, though there was some cross-contamination between fractions. Differential centrifugation produces a rough fractionation that can be further purified.

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    Download as docx, pdf, or txt
    4K views2 pages

    Bio 120 Ex 3

    Uploaded by

    piiish
    In the experiment, mungbean seeds were germinated in the dark to reduce photosynthesis and lessen starch accumulation. Differential centrifugation was used to separate subcellular components of the mungbean crude extract based on size and density. Chemical tests showed that particles separated into pellet I (PI) contained mostly starch and nucleic acids, pellet II (PII) contained lipids and nucleic acids, and supernatant II (SII) contained oxidative particles, nucleic acids, and proteins. The results mostly aligned with expectations, though there was some cross-contamination between fractions. Differential centrifugation produces a rough fractionation that can be further purified.

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    In the experiment, mungbean seeds had to be germinated in the dark to reduce photosynthesis and lessen starch
    accumulation.The mungbean crude extract was made by homogenizing the mungbean tissue with phosphate buffer in a
    blender. Only the epicotyl and hypocotyl of the sprouts were taken because these contain the actual cells of the mungbean.
    Cold phosphate buffer was used in order to stop enzymatic reactions, prevent the cell organellles from bursting, and stop
    pH changes (Huber et. al., 2003). The homogenate was then filtered through cheesecloth to remove insoluble tissues
    (mrothery.co.uk). The particles varying in size are then separated using centrifugation at different speeds (Lehninger, et.
    al., 2004) . The centrifuge was operated with two tubes of equal weight placed on opposite sides of the rotor so that it
    would be balanced while the machine is spinning.
    The chemical tests are used to determine the organelles present in each suspension. I2KI tests the presence of
    starch, Sudan IV tests the presence of lipids, Janus Green tests the presence of oxidative particles (such as mitochondria),
    acetocarmine tests for the presence of nucleic acids, and the biuret test is for determining the presence of proteins (Kirby,
    1950) (Science and Health Education Partnership) (Ghazi-Khansari, 2007).
    Prior to centrifugation, the crude extract was subjected to the given chemical tests. It was found that it had several
    stained structures in all the tests, most of all in the I2KI, acetocarmine, and biuret tests. This observation accords with the
    theoretical result, which is that the crude extract would contain high amounts of each type of particle. Upon the first round
    of centrifugation, separation of particles occurred, thus particles in the first pellet would theoretically not be found in the
    first supernatant. The test results were that there were high amounts of all particles in the SI while there was a high amount
    of starch followed by nucleic acids in the PI. According to Lehninger, et. al. (2004), PI contains whole cells, nuclei,
    cytoskeletons, and plasma membrane. SI on the other hand would contain the rest of the cell components. After
    centrifugation of SI, PII was separated. The results showed that PII contained mostly lipids and nucleic acids. This is not
    consistent with reference, which explains that PII contains large particles such as lysosomes, microbodies, and
    mitochondria, some of which are also oxidative . It is expected that PII then would have an abundance of stained structures
    upon application of Janus Green. This result was nonetheless reflected by SII, containing high amounts of oxidative
    particles, nucleic acids, and proteins. However, it has been noted in other sources that there is cross contamination between
    the PII and the consequent PIII, meaning that mitochondria show up in PIII and lysosomes appear in PII (YCMOU
    Elearning Drive, 2002). Thus the results are valid, since lysosomes may contain lipids and nucleic acids (as reflective of
    the relatively high amounts of Sudan IV- and acetocarmine-stained particles in PII) still being digested, and SII contained a
    high amount of oxidative particles.
    Based on the principle of differential centrifugation, the particles were isolated by sedimentation. The heavier
    particles settle first, then there is a gradual separation of the lighter particles. Thus, there are larger particles found in SI and
    PI than in SII and PII. This principle is also reflected in the distribution of starch, which is a large molecule composed of
    many glucose units (Berg, et. al., 2002). More starch molecules accumulated in SI and PI than in SII and PII.
    Water-soluble enzymes are found in the cytosol and are found in abundance in SII due to its small density and
    solubility. DNA, on the other hand, was sedimented in PI because of its relatively high density. Below is a table recounting
    the subcellular components and the fraction that they can be found in abundance, as well as the reason for their
    accumulation.

    Table 1. The evidences or bases of the occurrence of different subcellular components in the fractions.
    Subcellular components Fraction Evidence/basis
    Plasma membrane PI Present in large amounts, relatively large molecular structure
    Nucleus PI High number of stained structures by acetocarmine, large molecular structure
    Ribosomes SII High degree of hue in biuret test, small molecular structure
    Membranes of organelles SI High number of stained structures by
    Mitochondria SII High number of stained structures by Janus Green
    Soluble enzymes SII Small molecular structure, cannot be sedimented by centrifugation due to solubility
    Starch granules PI High number of stained structures by I2KI, large molecular structure
    Water SII Cannot be sedimented
    Salts SII Small molecular structure

    Differential centrifugation produces only a rough fractionation of cellular components, and it is usually purified further by
    density-gradient centrifugation. A limitation of the use of differential centrifugation is that particles with similar weights
    and densities albeit different in nature will not be isolated.
    References:

    Alkari, S. (2007) Centrifugation. Online Counseling Resource: YCMOU Elearning Drive. Yashwantrao Chavan
    Maharashtra Open University, Nashik.

    Berg, J. M., L. Stryer, J. L. Tymoczko (2002). Biochemistry, 5th ed. Retrieved from
    http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=stryer∂=A1517#A1522

    Ghazi-Khansari, M., A. Mohammadi-Bardbori, and M-J. Hosseini (2007). Using Janus Green B to study paraquat toxicity
    in rat liver mitochondria. Abstract retrieved from Annals of the New York Academy of Sciences database. doi: 10.1196

    Kirby, H. (1950). Materials and methods in the study of protozoa. Retrieved from http://books.google.com

    Lehninger, A. L., M. M. Cox, and D. L. Nelson (2004). Principles of Biochemistry. W.H. Freeman & Company.

    Science and Health Education Partnership. Testing of lipids, proteins, and carbohydrates. Retrieved from
    http://www.seplessons.org/node/362

    Rothery, M. Techniques. Retrieved from http://www.mrothery.co.uk/module1/Mod%201%20techniques.htm

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