In the experiment, mungbean seeds had to be germinated in the dark to reduce ph otosynthesis and lessen starch accumulation.The accumulation.The mungbean crude extract was made by homogenizing the mungbean tissue with phosphate buffer in a blender. Only the epicotyl and h ypocotyl ypocotyl of the t he sprouts were taken because these contain the actual cells of the mungbean. Cold phosphate buffer was used in order to stop enzymatic reactions, reactions, prevent the cell organellles from bursting, and stop pH changes (Huber et. al., 2003). Th e 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 i s 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 lip ids, Janus Green tests the presence of oxidative particles (such a s mitochondria), acetocarmine tests for the presence of nucleic a cids, and the biuret test is for determining the presence of proteins (Kirby, 1950) (Science and Health Education Partnership) (Ghazi-Khansari, (Ghazi-Khansari, 2007). Prior to centrifugation, the crude extract was subjected to the given chemical tests. It was found that it ha d several stained structures in all the tests, most of a ll in the I2KI, a cetocarmine, cetocarmine, 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 th ere 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 th e cell components. After centrifugation of SI, PII was separated. The results showed that PII contained mostly lipids and n ucleic 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 structures upon application of Janus Green. This result was n onetheless reflected 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 th e 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 an d nucleic acids (as reflective of the relatively high amounts of Sudan IV- and acetocarmine-stained particles in in PII) still being digested, and SII contained a high amount of oxidative particles. Based on the principle of di fferential centrifugation, centrifugation, the particles were isolated by sedimentation. sedimentation. The heavier particles settle first, then there is a gradual separation 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 enzymes are found in th e cytosol cytosol and are found in abundance in SII due to its small density and solubility. 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 th ey can be found in abundance, as well as the r eason 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
Nucleus
Ribosomes
Present in large amounts, relatively large molecular structure PI
SII
High number of stained structures by acetocarmine, large molecular structure 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 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. Centrifugation. Online Counseling Resource: YCMOU Elearning Drive. Yashwantrao Chavan Maharashtra Open University, Nashik. Berg, J. M., L. Stryer, J. L. Tymoczko (2002). Biochemistry, Biochemistry, 5th ed. Retrieved from http://www.ncbi.nlm.nih.gov http://www .ncbi.nlm.nih.gov/bookshelf/br.fcgi? /bookshelf/br.fcgi?book=stryer=A1517#A1522 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. Partnership. Testing of lipids, proteins, and carbohydrates. Retrieved from http://www.seplessons.org/node/362 Rothery, M. Techniques. Retrieved from http://www http://www.mrothery.co.uk/module1/Mo .mrothery.co.uk/module1/Mod%201%20techniques d%201%20techniques.htm .htm
